mbinfo MBInfo http://www.mechanobio.info/ http://www.mechanobio.info/ demali-2002-3 Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot DeMali K.A., Barlow C.A., Burridge K. The pull down assays between vinculin and the Arp2/3 complex, involving crude platelet extracts [Figs 6B, 6C, 6D(of p34)] could not be curated since the source of the platelets could not be identified. kdemali@med.unc.edu J. Cell Biol. (0021-9525) 2002 Only protein-protein interactions imex curation demali-2002-1 Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion. human-a_431 Homo sapiens epidermoid carcinoma a_431 Human epidermoid carcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot DeMali K.A., Barlow C.A., Burridge K. The pull down assays between vinculin and the Arp2/3 complex, involving crude platelet extracts [Figs 6B, 6C, 6D(of p34)] could not be curated since the source of the platelets could not be identified. kdemali@med.unc.edu J. Cell Biol. (0021-9525) 2002 Only protein-protein interactions imex curation demali-2002-2 Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion. human-hs68 Homo sapiens Hs68 normal diploid foreskin fibroblasts hs68 Human Hs68 normal diploid foreskin fibroblasts anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot DeMali K.A., Barlow C.A., Burridge K. The Hs68 cells were allowed to spread on Fibronectin-coated coverslips by centrifuging the cells onto the coverslips, followed by lysis of the cells and immunoprecipitation of vinculin. The pull down assays between vinculin and the Arp2/3 complex, involving crude platelet extracts [Figs 6B, 6C, 6D(of p34)] could not be curated since the source of the platelets could not be identified. kdemali@med.unc.edu J. Cell Biol. (0021-9525) 2002 Only protein-protein interactions imex curation demali-2002-4 Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion. mouse-embryonic_fibroblast Mus musculus embryonic fibroblasts embryonic_fibroblast Embryonic fibroblast Fibroblasts taken from an embryonic as opposed to a foetal or adult source. Foetus is defined as the stage at which tissue diferentiation is initiated. Tissue taken from an embryonic as opposed to a foetal or adult source. Embryo is defined as the development stage prior to organ formation. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot DeMali K.A., Barlow C.A., Burridge K. The pull down assays between vinculin and the Arp2/3 complex, involving crude platelet extracts [Figs 6B, 6C, 6D(of p34)] could not be curated since the source of the platelets could not be identified. kdemali@med.unc.edu J. Cell Biol. (0021-9525) 2002 Only protein-protein interactions imex curation ponsin_canfa_protein Ponsin protein protein canlf Canis familiaris (dog) Canis lupus familiaris Dog Canis familiaris p12003-1 Vinculin Vinculin VCL VINC1 Metavinculin protein protein chick Gallus gallus (Chicken) Chicken MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVSAVQAAVSNLVRVGKETVQTTEDQILKRDMPPAFIKVENACTKLVRAAQMLQADPYSVPARDYLIDGSRGILSGTSDLLLTFDEAEVRKIIRVCKGILEYLTVAEVVETMEDLVTYTKNLGPGMTKMAKMIDERQQELTHQEHRVMLVNSMNTVKELLPVLISAMKIFVTTKNTKSQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWDEDAWASKDTEAMKRALALIDSKMNQAKGWLRDPNAPPGDAGEQAIRQILDEAGKAGELCAGKERREILGTCKTLGQMTDQLADLRARGQGATPMAMQKAQQVSQGLDLLTAKVENAARKLEAMTNSKQAIAKKIDAAQNWLADPNGGSEGEEHIRGIMSEARKVAELCEEPKERDDILRSLGEISALTAKLSDLRRHGKGDSPEARALAKQIATSLQNLQSKTNRAVANTRPVKAAVHLEGKIEQAQRWIDNPTVDDRGVGQAAIRGLVAEGRRLANVMMGPYRQDLLAKCDRVDQLAAQLADLAARGEGESPQARAIAAQLQDSLKDLKARMQEAMTQEVSDVFSDTTTPIKLLAVAATAPSDTPNREEVFEERAANFENHAARLGATAEKAAAVGTANKTTVEGIQATVKSARELTPQVVSAARILLRNPGNQAAYEHFETMKNQWIDNVEKMTGLVDEAIDTKSLLDASEEAIKKDLDKCKVAMANMQPQMLVAGATSIARRANRILLVAKREVENSEDPKFREAVKAASDELSKTISPMVMDAKAVAGNISDPGLQKSFLDSGYRILGAVAKVREAFQPQEPDFPPPPPDLEHLHLTDELAPPKPPLPEGEVPPPRPPPPEEKDEEFPEQKAGEAINQPMMMAARQLHDEARKWSSKGNDIIAAAKRMALLMAEMSRLVRGGSGNKRALIQCAKDIAKASDEVTRLAKEVAKQCTDKRIRTNLLQVCERIPTISTQLKILSTVKATMLGRTNISDEESEQATEMLVHNAQNLMQSVKETVREAEAASIKIRTDAGFTLRWVRKTPWYQ 6F585201260BA47F vinex_mouse Vinexin Sorbs3 Scam1 Sh3d4 SH3 domain-containing protein SH3P3 SH3-containing adapter molecule 1 Sorbin and SH3 domain-containing protein 3 protein protein mouse Mus musculus Mouse MARILGVGRSSASSLNNKEDNESDVALLSPKDPNRVHTKEQLAHPASSNLDPSMQGLPAGLSLDDFIPGHLRTHIGSSSRGTRVPVIRNGGSNTLNFQFHDPAPRTVCNGCPPPRRDGSLNPDPAWYQTWPGPGSRPSMSPKPPASQHAQNWSATWTKDSKRQDKRWVKYEGIGPVDESGMPIAPRSSVDSPRDWYRRMFQQIHRKMPDLQLDWTLEDPPKVVSARASSAEPRHLGTLQRPASRPGTTETSSGRNWNHSEETSRNTFNYNFRPSSSGLHPPNQVPRHREKVENVWTEDSWNQFLHELETGHKPKKPLVDDPVEKPAQPIEVLLERELAKLSAELDKDLRAIETRLPSPKNSQAPRRPLEQPGLEQQPSARLSSAWRPNSPHAPYFSSSRPLSPHRMADGGGSPFLGRRDFVYPSSAREPSASERGSSPSRKEEKKRKAARLKFDFQAQSPKELSLQKGDIVYIHKEVDKNWLEGEHHGRLGIFPANYVEVLPADEIPKPIKPPTYQVLEYGDAVAQYTFKGDLEVELSFRKGERICLIRKVNEHWYEGRITGTGRQGIFPASYVQINREPRLRLCDDGPQLPASPNPTTTAHLSSHSHPSSIPVDPTDWGGRTSPRRSAFPFPITLQEPRSQTQSLNTPGPTLSHPRATSRPINLGPSSPNTEIHWTPYRAMYQYRPQNEDELELREGDRVDVMQQCDDGWFVGVSRRTQKFGTFPGNYVAPV vasp_human Vasodilator-stimulated phosphoprotein VASP protein protein human Homo sapiens Human MSETVICSSRATVMLYDDGNKRWLPAGTGPQAFSRVQIYHNPTANSFRVVGRKMQPDQQVVINCAIVRGVKYNQATPNFHQWRDARQVWGLNFGSKEDAAQFAAGMASALEALEGGGPPPPPALPTWSVPNGPSPEEVEQQKRQQPGPSEHIERRVSNAGGPPAPPAGGPPPPPGPPPPPGPPPPPGLPPSGVPAAAHGAGGGPPPAPPLPAAQGPGGGGAGAPGLAAAIAGAKLRKVSKQEEASGGPTAPKAESGRSGGGGLMEEMNAMLARRRKATQVGEKTPKDESANQEEPEARVPAQSESVRRPWEKNSTTLPRMKSSSSVTTSETQPCTPSSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGSP arpc2_human Actin-related protein 2/3 complex subunit 2 ARPC2 ARC34 PRO2446 Arp2/3 complex 34 kDa subunit protein protein human Homo sapiens Human MILLEVNNRIIEETLALKFENAAAGNKPEAVEVTFADFDGVLYHISNPNGDKTKVMVSISLKFYKELQAHGADELLKRVYGSFLVNPESGYNVSLLYDLENLPASKDSIVHQAGMLKRNCFASVFEKYFQFQEEGKEGENRAVIHYRDDETMYVESKKDRVTVVFSTVFKDDDDVVIGKVFMQEFKEGRRASHTAPQVLFSHREPPLELKDTDAAVGDNIGYITFVLFPRHTNASARDNTINLIHTFRDYLHYHIKCSKAYIHTRMRAKTSDFLKVLNRARPDAEKKEMKTITGKTFSSR vinc_human Vinculin Metavinculin VCL protein protein human Homo sapiens Human MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKETVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTSDLLLTFDEAEVRKIIRVCKGILEYLTVAEVVETMEDLVTYTKNLGPGMTKMAKMIDERQQELTHQEHRVMLVNSMNTVKELLPVLISAMKIFVTTKNSKNQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWDEDAWASKDTEAMKRALASIDSKLNQAKGWLRDPSASPGDAGEQAIRQILDEAGKVGELCAGKERREILGTCKMLGQMTDQVADLRARGQGSSPVAMQKAQQVSQGLDVLTAKVENAARKLEAMTNSKQSIAKKIDAAQNWLADPNGGPEGEEQIRGALAEARKIAELCDDPKERDDILRSLGEISALTSKLADLRRQGKGDSPEARALAKQVATALQNLQTKTNRAVANSRPAKAAVHLEGKIEQAQRWIDNPTVDDRGVGQAAIRGLVAEGHRLANVMMGPYRQDLLAKCDRVDQLTAQLADLAARGEGESPQARALASQLQDSLKDLKARMQEAMTQEVSDVFSDTTTPIKLLAVAATAPPDAPNREEVFDERAANFENHSGKLGATAEKAAAVGTANKSTVEGIQASVKTARELTPQVVSAARILLRNPGNQAAYEHFETMKNQWIDNVEKMTGLVDEAIDTKSLLDASEEAIKKDLDKCKVAMANIQPQMLVAGATSIARRANRILLVAKREVENSEDPKFREAVKAASDELSKTISPMVMDAKAVAGNISDPGLQKSFLDSGYRILGAVAKVREAFQPQEPDFPPPPPDLEQLRLTDELAPPKPPLPEGEVPPPRPPPPEEKDEEFPEQKAGEVINQPMMMAARQLHDEARKWSSKPGIPAAEVGIGVVAEADAADAAGFPVPPDMEDDYEPELLLMPSNQPVNQPILAAAQSLHREATKWSSKGNDIIAAAKRMALLMAEMSRLVRGGSGTKRALIQCAKDIAKASDEVTRLAKEVAKQCTDKRIRTNLLQVCERIPTISTQLKILSTVKATMLGRTNISDEESEQATEMLVHNAQNLMQSVKETVREAEAASIKIRTDAGFTLRWVRKTPWYQ arpc2_mouse Actin-related protein 2/3 complex subunit 2 Arp2/3 complex 34 kDa subunit Arpc2 protein protein mouse Mus musculus Mouse MILLEVNNRIIEETLALKFENAAAGNKPEAVEVTFADFDGVLYHISNPNGDKTKVMVSISLKFYKELQAHGADELLKRVYGSFLVNPEPGYNVSLLYDLENLPASKDSIVHQAGMLKRNCFASVFEKYFQFQEEGKEGENRAVIHYRDDETMYVESKKDRVTVVFSTVFKDDDDVVIGKVFMQEFKEGRRASHTAPQVLFSHREPPLELKDTDAAVGDNIGYITFVLFPRHTNATARDNTINLIHTFRDYLHYHIKCSKAYIHTRMRAKTSDFLKVLNRARPDAEKKEMKTITGKTFSSR vcl-ponsin 1388781 n/a 1388785 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 n/a 1388786 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 6D (second panel). The vinculin [811-881] fragments with point mutations P876A and P878A also interacted with ponsin. vcl-sorbs3-1 1388781 n/a 1388786 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) n/a 1388787 unspecified role unspecified role prey prey mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast physical association physical association false false false Fig 6D (third panel). The vinculin [811-881] fragments with point mutations P876A and P878A also interacted with vinexin. vcl-vasp-4 1388781 n/a 1388788 unspecified role unspecified role prey prey human-a_431 Homo sapiens epidermoid carcinoma a_431 Human epidermoid carcinoma n/a 1388786 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 6D (fourth panel). The vinculin [811-881] fragments with point mutations P876A and P878A also interacted with VASP. vcl-arpc2-7 1388781 n/a 1388789 unspecified role unspecified role prey prey human-platelet Homo sapiens platelet, thrombocytes platelet Platelet, thrombocyte n/a 1388786 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig 7. vcl-arpc2-1 1388782 n/a 1388790 unspecified role unspecified role bait bait n/a 1388789 unspecified role unspecified role prey prey physical association physical association false false false Fig 1A, 2, 3A, 3B, 4. Vinculin interacted with the Arp2/3 complex only when the A431 cells were stimulated with EGF. The interaction was noticed 30 sec after EGF stimulation and decreased 15-20 min after the EGF stimulation (Fig 1A). Vinculin did not interact with the Arp2/3 complex in Wortmannin (PI3K inhibitor)-treated cells (Fig 2). Vinculin interacted with the Arp2/3 complex in the presence of constitutively active Rac (Fig 3B), but not in the presence of dominant negative Rac (Fig 3A). The interaction of vinculin with the Arp2/3 complex was slightly increased in the presence of PIP2, increased more in the presence of GST-VCA (of Wave protein) and was highest in the presence of both PIP2 and GST-VCA (Fig 4). vcl-arpc2-3 1388783 n/a 1388789 unspecified role unspecified role prey prey n/a 1388790 unspecified role unspecified role bait bait physical association physical association false false false Fig 1C. The interaction of vinculin with the Arp2/3 complex was low in stationary cells, but increased in cells allowed to spread on FN-coated coverslips. vcl-arpc2-5 1388784 n/a 1388791 unspecified role unspecified role prey prey n/a 1388786 unspecified role unspecified role bait bait P12003-1:p.Pro878Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig 6E. Vinculin WT interacts with the Arp2/3 complex only in FBS-stimulated cells. Vinculin[P878A] did not interact with the Arp2/3 complex in the presence or absence of FBS stimulation.