IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. human-sw13 human sw13 cells from bone sw13 SW1353 Species: human, Caucasian female 72 years old; Tissue: bone; Tumor: chondrosarcoma fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. in vitro In vitro cosedimentation cosedimentation predetermined predetermined participant predetermined Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip western blot western blot Immuno blot Immuno blot Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. human-t24 Homo sapiens T24 bladder carcinoma cells t24 Human bladder carcinoma fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. (0021-9525) 2001 Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Inada H., Izawa I., Nishizawa M., Fujita E., Kiyono T., Takahashi T., Momoi T., Inagaki M. Apoptosis - Interactions involving proteins with a function related to apoptosis minagaki@aichi-cc.jp Accepted 2007-OCT-4 by JYOTI. 12-Oct-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Cell Biol. (0021-9525) 2001 tradd_human Tumor necrosis factor receptor type 1-associated DEATH domain protein TRADD TNFRSF1A-associated via death domain protein protein human Homo sapiens Human MAAGQNGHEEWVGSAYLFVESSLDKVVLSDAYAHPQQKVAVYRALQAALAESGGSPDVLQMLKIHRSDPQLIVQLRFCGRQPCGRFLRAYREGALRAALQRSLAAALAQHSVPLQLELRAGAERLDALLADEERCLSCILAQQPDRLRDEELAELEDALRNLKCGSGARGGDGEVASAPLQPPVPSLSEVKPPPPPPPAQTFLFQGQPVVNRPLSLKDQQTFARSVGLKWRKVGRSLQRGCRALRDPALDSLAYEYEREGLYEQAFQLLRRFVQAEGRRATLQRLVEALEENELTSLAEDLLGLTDPNGGLA DIP protein Q15628 original sequence version: 92 DIP protein Q15628 original sequence version: 132 DIP protein Q15628 original sequence version: 156 DIP protein Q15628 original sequence version: 77 5645D7E63E5FF05A k2c8_human Keratin, type II cytoskeletal 8 Keratin-8 KRT8 CYK8 Cytokeratin-8 Type-II keratin Kb8 protein protein human Homo sapiens Human MSIRVTQKSYKVSTSGPRAFSSRSYTSGPGSRISSSSFSRVGSSNFRGGLGGGYGGASGMGGITAVTVNQSLLSPLVLEVDPNIQAVRTQEKEQIKTLNNKFASFIDKVRFLEQQNKMLETKWSLLQQQKTARSNMDNMFESYINNLRRQLETLGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTEMENEFVLIKKDVDEAYMNKVELESRLEGLTDEINFLRQLYEEEIRELQSQISDTSVVLSMDNSRSLDMDSIIAEVKAQYEDIANRSRAEAESMYQIKYEELQSLAGKHGDDLRRTKTEISEMNRNISRLQAEIEGLKGQRASLEAAIADAEQRGELAIKDANAKLSELEAALQRAKQDMARQLREYQELMNVKLALDIEIATYRKLLEGEESRLESGMQNMSIHTKTTSGYAGGLSSAYGGLTSPGLSYSLGSSFGSGAGSSSFSRTSSSRAVVVKKIETRDGKLVSESSDVLPK DIP protein P05787 original sequence version: 186 DIP protein P05787 original sequence version: 146 B0BC730B65929D37 tnr1a_human Tumor necrosis factor receptor superfamily member 1A TNFRSF1A TNFAR TNFR1 p60 Tumor necrosis factor receptor 1 Tumor necrosis factor receptor type I p55 protein protein human Homo sapiens Human MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQKWEDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR DIP protein P19438 original sequence version: 135 DIP protein P19438 original sequence version: 150 DIP protein P19438 original sequence version: 177 4CEFBA96D03B8225 k1c14_human Keratin, type I cytoskeletal 14 Cytokeratin-14 KRT14 Keratin-14 protein protein human Homo sapiens Human MTTCSRQFTSSSSMKGSCGIGGGIGGGSSRISSVLAGGSCRAPSTYGGGLSVSSSRFSSGGACGLGGGYGGGFSSSSSSFGSGFGGGYGGGLGAGLGGGFGGGFAGGDGLLVGSEKVTMQNLNDRLASYLDKVRALEEANADLEVKIRDWYQRQRPAEIKDYSPYFKTIEDLRNKILTATVDNANVLLQIDNARLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLARADLEMQIESLKEELAYLKKNHEEEMNALRGQVGGDVNVEMDAAPGVDLSRILNEMRDQYEKMAEKNRKDAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLEETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGEDAHLSSSQFSSGSQSSRDVTSSSRQIRTKVMDVHDGKVVSTHEQVLRTKN DIP protein P02533 original sequence version: 143 120BA30BA2F8E397 k1c18_human Keratin, type I cytoskeletal 18 KRT18 CYK18 Keratin-18 PIG46 Cytokeratin-18 Cell proliferation-inducing gene 46 protein protein protein human Homo sapiens Human MSFTTRSTFSTNYRSLGSVQAPSYGARPVSSAASVYAGAGGSGSRISVSRSTSFRGGMGSGGLATGIAGGLAGMGGIQNEKETMQSLNDRLASYLDRVRSLETENRRLESKIREHLEKKGPQVRDWSHYFKIIEDLRAQIFANTVDNARIVLQIDNARLAADDFRVKYETELAMRQSVENDIHGLRKVIDDTNITRLQLETEIEALKEELLFMKKNHEEEVKGLQAQIASSGLTVEVDAPKSQDLAKIMADIRAQYDELARKNREELDKYWSQQIEESTTVVTTQSAEVGAAETTLTELRRTVQSLEIDLDSMRNLKASLENSLREVEARYALQMEQLNGILLHLESELAQTRAEGQRQAQEYEALLNIKVKLEAEIATYRRLLEDGEDFNLGDALDSSNSMQTIQKTTTRRIVDGKVVSETNDTKVLRH DIP protein P05783 original sequence version: 186 DIP protein P05783 original sequence version: 175 1E5604C6BCC7A17A tradd-krt18 6 10 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind full dna sequence full identification by DNA sequencing full dna sequence certain certain sequence position certain certain certain sequence position certain 14 unspecified role unspecified role bait bait physical association physical association Both Krt8 and Krt18 were used as bait on the human liver cDNA library, but only Krt18 picked up the Tradd c-terminal fragment. To identify proteins interacting with K8/18, we screened a human liver cDNA library, using the yeast two-hybrid procedure with full-length K8 or K18 as a bait. Figure 1A. tradd-krt18-1 8 14 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind predetermined featur predetermined feature predetermined featur certain certain sequence position certain certain certain sequence position certain physical association physical association Both the c-terminal and full-length Tradd were able to interact with Krt18 but there was strongest interaction with the Tradd c-terminal fragment. Figure 1B To confirm that full-length TRADD (TRADD-F) interacts with K18 and to determine if TRADD can bind to other IF proteins, we examined the interaction of TRADD-F and TRADD-C with K5, K8, K14, K18, and type III IF proteins in the two-hybrid system (Fig. 1 B). tradd-krt14 8 13 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind predetermined featur predetermined feature predetermined featur certain certain sequence position certain certain certain sequence position certain physical association physical association Both the c-terminal and full-length Tradd were able to interact with Krt14 but there was strongest interaction with the Tradd c-terminal fragment. Figure 1B To confirm that full-length TRADD (TRADD-F) interacts with K18 and to determine if TRADD can bind to other IF proteins, we examined the interaction of TRADD-F and TRADD-C with K5, K8, K14, K18, and type III IF proteins in the two-hybrid system (Fig. 1 B). tradd-krt18-2 8 14 unspecified role unspecified role bait bait n-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind predetermined featur predetermined feature predetermined featur certain certain sequence position certain certain certain sequence position certain physical association physical association The coil Ia region of the rod domain of Krt18 specifically interacted with both full-length TRADD (TRADD-F) and the c-terminal region of TRADD (TRADD-C). Figure 1C We next analyzed the region of K18 that contained the TRADD interaction site. For this, the binding between a series of truncations of K18 and TRADD-F or TRADD-C was determined using the two-hybrid system (Fig. 1 C). krt8-krt18-3 4 14 unspecified role unspecified role neutral component neutral component in vitro In vitro 11 unspecified role unspecified role neutral component neutral component in vitro In vitro physical association physical association Krt18 assembled with K8 independantly of GST or GST-Tradd. Figure 2A, 2B Incubation in filament assembly buffer induced a rapid polymerization of K8 and K18 (Fig. 2A, lanes 1). As a control, recombinant GST was not sedimented in the presence of K8/18 (Fig. 2 A and B, lane 3). tradd-krt18-3 4 10 unspecified role unspecified role neutral component neutral component region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component in vitro In vitro 11 unspecified role unspecified role neutral component neutral component in vitro In vitro physical association physical association Because Tradd interacted with K18, but not with K8, in the two-hybrid analysis, the Tradd cosedimention with the K8/18 polymer via direct binding to K18. In addition, Tradd did not sediment with vimentin. Figure 2A, 2B In the absence of K8/18, GST�TRADD was not sedimented (Fig. 2 A and B, lane 4), but in the presence of K8/18, a substantial portion of GST�TRADD was precipitated (Fig. 2 A and B, lane 5). Because TRADD interacted with K18, but not with K8 in the two-hybrid analysis, the TRADD sedimenting with K8/18 was thought to be associated with the K8/18 polymer via direct binding to K18. In addition, TRADD did not sediment with vimentin (Fig. 2 A and B, lane 6) or desmin (Fig. 2 A and B, lane 7), findings consistent with results obtained using the two-hybrid method. tradd-krt18-4 9 14 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 colocalization colocalization HeLa cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody. Overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments. Figure 3A In attempts to characterize the K18�TRADD association in vivo, we first determined the subcellular localization of overexpressed myc-tagged TRADD in various epithelial cells. We transfected HeLa cells, T24 cells, MDCK cells, and immortalized human mammary epithelial cells (HMEC) with a myc-tagged vector encoding the full-length TRADD. 16 h later, the cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody (Fig. 3A). In various cells, overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments (Fig. 3 A). tradd-krt18-5 7 14 unspecified role unspecified role prey prey human-t24 Homo sapiens T24 bladder carcinoma cells t24 Human bladder carcinoma 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-t24 Homo sapiens T24 bladder carcinoma cells t24 Human bladder carcinoma colocalization colocalization T24 cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody. Overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments. Figure 3A In attempts to characterize the K18�TRADD association in vivo, we first determined the subcellular localization of overexpressed myc-tagged TRADD in various epithelial cells. We transfected HeLa cells, T24 cells, MDCK cells, and immortalized human mammary epithelial cells (HMEC) with a myc-tagged vector encoding the full-length TRADD. 16 h later, the cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody (Fig. 3A). In various cells, overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments (Fig. 3 A). tradd-krt18-6 2 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 14 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line colocalization colocalization MDCK cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody. Overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments. Figure 3A In attempts to characterize the K18�TRADD association in vivo, we first determined the subcellular localization of overexpressed myc-tagged TRADD in various epithelial cells. We transfected HeLa cells, T24 cells, MDCK cells, and immortalized human mammary epithelial cells (HMEC) with a myc-tagged vector encoding the full-length TRADD. 16 h later, the cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody (Fig. 3A). In various cells, overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments (Fig. 3 A). tradd-krt18-7 3 14 unspecified role unspecified role prey prey human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium colocalization colocalization In immortalized human mammary epithelial cells (HMEC) myc�TRADD filaments markedly colocalized with K8/18 filaments. Figure 3A, 4C, 5A, 6A In attempts to characterize the K18�TRADD association in vivo, we first determined the subcellular localization of overexpressed myc-tagged TRADD in various epithelial cells. We transfected HeLa cells, T24 cells, MDCK cells, and immortalized human mammary epithelial cells (HMEC) with a myc-tagged vector encoding the full-length TRADD. 16 h later, the cells were fixed and doubly stained with anti-myc polyclonal antibody and anti-K18 monoclonal antibody (Fig. 3A). In various cells, overexpressed myc�TRADD exhibited a filamentous pattern, and these myc�TRADD filaments markedly colocalized with K8/18 filaments (Fig. 3 A). To determine the intracellular distribution of TRADD and to check if TRADD colocalizes with K8/18 filament networks, HMEC were fixed with 50% methanol/50% acetone and double stained with anti-TRADD polyclonal antibody and anti-K18 monoclonal antibody. TRADD was present in the cytoplasm and showed some filamentous patterns, and there was a significant overlap between filamentous TRADD and K18 (Fig. 4 C, a-c). To further confirm the colocalization of TRADD with K18, HMEC cells were transfected with a K8 deletion mutant to disrupt K8/18 filaments. Cells expressing the myc-tagged K8 deletion mutant (aa 1-239 of K8) showed disorganization of K8/18 filaments and the disappearance of the filamentous distribution of TRADD. These results confirm the colocalization of TRADD with K8/18 filaments, and suggest that K8/18 filament organization affects the subcellular distribution of TRADD. Since TRADD is a TNFR1 adaptor protein recruited to the TNFR1 in a TNF-dependent manner, we next determined if TNF stimulation would affect K18-TRADD association. To observe effects of TNF treatment on the subcellular distribution of TRADD, HMEC were untreated or incubated for 20 min with increasing concentrations of TNF, and then doubly stained for TRADD and K18 (Fig. 5 A). At the physiological concentration of TNF (3 ng/ml), there were no apparent changes in the colocalization of TRADD and K18. At 30 ng/ml of TNF, the filamentous pattern of TRADD distribution began to diminish (unpublished data). At the high dose of TNF (100 ng/ml), TRADD dissociated from K8/18 filaments, and dispersed diffusely (Fig. 5 A). On treatment with TNF, TRADD dissociated from K8/18 filaments, and dispersed diffusely in the cytoplasm. Other stimuli did not alter subcellular distribution of TRADD. To search for the role of K18-TRADD interaction in cellular responses to various apoptotic stimuli, we treated HMEC with 1microM aclarubicin (ACR), a topoisomerase inhibitor, 10microg/ml neocarzinostatin (NCS), a chemical that directly causes DNA breaks, 1microM taxol, a microtubule stabilizer, and UV irradiation (100 J/m2). 20 min after treatment with these stimuli, we examined the distribution of TRADD using immunostaining techniques (Fig. 6 A). tradd-krt18-8 5 10 unspecified role unspecified role bait bait human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium 14 unspecified role unspecified role prey prey human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium physical association physical association K18 was coimmunoprecipitated with TRADD, using the anti-TRADD antibody. This coprecipitation was barely visible in the 100 ng/ml TNF-treated HMEC samples suggesting that TRADD is specifically dissociated from K18 by TNF. Figure 4B, 5B, 6B Using the rabbit anti-TRADD antibody, we precipitated complexes containing TRADD proteins from 1% NP-40 soluble fraction of HMEC lysates (Fig. 4B). TRADD was immunoprecipitated with anti-TRADD antibody, but not with control rabbit IgG. K18 was coimmunoprecipitated with TRADD, using the anti-TRADD antibody. We next checked the binding of TRADD with K18 after TNF treatment by immunoprecipitation (Fig. 5 B). Lysates prepared from HMEC treated for 20 min with various doses of TNF, or left untreated, were immunoprecipitated with anti-TRADD antibody, and the presence of K18 or TNFR1 in the TRADD immune complex was examined. Coprecipitating K18 was readily detected in lysates from untreated (control) cells and physiological dose TNF (3 ng/ml)-treated cells but was barely visible in the 100 ng/ml TNF-treated samples. We also checked TRADD-K18 binding after these treatments using immunoprecipitation methods (Fig. 6 B). tradd-tradd-3 5 10 unspecified role unspecified role prey prey human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium 10 unspecified role unspecified role bait bait human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium physical association physical association TRADD was coimmunoprecipitated with TRADD, using the anti-TRADD antibody. Figure 4B Using the rabbit anti-TRADD antibody, we precipitated complexes containing TRADD proteins from 1% NP-40 soluble fraction of HMEC lysates (Fig. 4B). TRADD was immunoprecipitated with anti-TRADD antibody, but not with control rabbit IgG. K18 was coimmunoprecipitated with TRADD, using the anti-TRADD antibody. tradd-krt18-9 1 14 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-sw13 human sw13 cells from bone sw13 SW1353 Species: human, Caucasian female 72 years old; Tissue: bone; Tumor: chondrosarcoma 10 unspecified role unspecified role neutral component neutral component human-sw13 human sw13 cells from bone sw13 SW1353 Species: human, Caucasian female 72 years old; Tissue: bone; Tumor: chondrosarcoma 11 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-sw13 human sw13 cells from bone sw13 SW1353 Species: human, Caucasian female 72 years old; Tissue: bone; Tumor: chondrosarcoma colocalization colocalization In K8/ 18-transfected SW13 cells, TRADD was recruited to overexpressed K8/18 filaments Figure 7C In untransfected SW13 cells, which are devoid of keratins, native TRADD was diffusely present in the cytoplasm (Fig. 7 C, a). In K8/ 18-transfected SW13 cells, TRADD was recruited to overexpressed K8/18 filaments (Fig. 7 C, d-f). In cells overexpressing the NH2 terminus of K18, TRADD markedly colocalized with the overexpressed NH2 terminus (Fig. 7 C, g-i). These results indicate that overexpressed K8/18 or K18 NH2 terminus could sequester endogenous TRADD, leading to the inhibition of TNF-induced apoptosis in SW13 cells. tradd-krt18-10 1 10 unspecified role unspecified role prey prey human-sw13 human sw13 cells from bone sw13 SW1353 Species: human, Caucasian female 72 years old; Tissue: bone; Tumor: chondrosarcoma 14 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminus sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain human-sw13 human sw13 cells from bone sw13 SW1353 Species: human, Caucasian female 72 years old; Tissue: bone; Tumor: chondrosarcoma colocalization colocalization In cells overexpressing the NH2 terminus of K18, TRADD markedly colocalized with the overexpressed NH2 terminus Figure 7C In untransfected SW13 cells, which are devoid of keratins, native TRADD was diffusely present in the cytoplasm (Fig. 7 C, a). In K8/ 18-transfected SW13 cells, TRADD was recruited to overexpressed K8/18 filaments (Fig. 7 C, d-f). In cells overexpressing the NH2 terminus of K18, TRADD markedly colocalized with the overexpressed NH2 terminus (Fig. 7 C, g-i). These results indicate that overexpressed K8/18 or K18 NH2 terminus could sequester endogenous TRADD, leading to the inhibition of TNF-induced apoptosis in SW13 cells. tradd-tnfr1 5 10 unspecified role unspecified role bait bait human-mammary epithelium Human mammary epithelium mammary_epithelium Mammary epithelium 12 unspecified role unspecified role prey prey physical association physical association The coprecipitation of K18 with TRADD was dissociated by TNF suggesting that TRADD binds with TNFR1. Figure 5B We next checked the binding of TRADD with K18 after TNF treatment by immunoprecipitation (Fig. 5 B). Lysates prepared from HMEC treated for 20 min with various doses of TNF, or left untreated, were immunoprecipitated with anti-TRADD antibody, and the presence of K18 or TNFR1 in the TRADD immune complex was examined. Coprecipitating K18 was readily detected in lysates from untreated (control) cells and physiological dose TNF (3 ng/ml)-treated cells but was barely visible in the 100 ng/ml TNF-treated samples.