MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. pla proximity ligation assay pELISA pELISA PLA anti tag immunost anti tag immunostaining anti tag immunost Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-m07e Homo sapiens hematopoietic cells m07e human hematopoietic cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. frap fluorescence recovery after photobleaching frap tag fluorescence tag visualisation by fluorescence tag fluorescence Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-m07e Homo sapiens hematopoietic cells m07e human hematopoietic cells pla proximity ligation assay pELISA pELISA PLA anti tag immunost anti tag immunostaining anti tag immunost Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro competition binding competition binding western blot western blot Immuno blot Immuno blot Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation An unexpected protein interaction promotes drug resistance in leukemia. An unexpected protein interaction promotes drug resistance in leukemia. Nature communications 2017 imex curation Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret predetermined predetermined participant predetermined Pitre A., Ge Y., Lin W., Wang Y., Fukuda Y., Temirov J., Phillips AH., Peters JL., Fan Y., Ma J., Nourse A., Sinha C., Lin H., Kriwacki R., Downing JR., Gruber TA., Centonze VE., Naren AP., Chen T., Schuetz JD. john.schuetz@stjude.org Accepted 2018-OCT-18 AT 09:53 BST AT 09:53 BST by LUANA Nature communications 2017 Only protein-protein interactions imex curation imex curation em55_human 55 kDa erythrocyte membrane protein Membrane protein, palmitoylated 1 MPP1 EMP55 DXS552E protein protein human Homo sapiens Human MTLKASEGESGGSMHTALSDLYLEHLLQKRSRPEAVSHPLNTVTEDMYTNGSPAPGSPAQVKGQEVRKVRLIQFEKVTEEPMGITLKLNEKQSCTVARILHGGMIHRQGSLHVGDEILEINGTNVTNHSVDQLQKAMKETKGMISLKVIPNQQSRLPALQMFMRAQFDYDPKKDNLIPCKEAGLKFATGDIIQIINKDDSNWWQGRVEGSSKESAGLIPSPELQEWRVASMAQSAPSEAPSCSPFGKKKKYKDKYLAKHSSIFDQLDVVSYEEVVRLPAFKRKTLVLIGASGVGRSHIKNALLSQNPEKFVYPVPYTTRPPRKSEEDGKEYHFISTEEMTRNISANEFLEFGSYQGNMFGTKFETVHQIHKQNKIAILDIEPQTLKIVRTAELSPFIVFIAPTDQGTQTEALQQLQKDSEAIRSQYAHYFDLSLVNNGVDETLKKLQEAFDQACSSPQWVPVSWVY DC68AA68EF48A26E mrp4_human ATP-binding cassette sub-family C member 4 ABCC4 MRP4 MRP/cMOAT-related ABC transporter Multi-specific organic anion transporter B MOATB Multidrug resistance-associated protein 4 protein protein human Homo sapiens Human MLPVYQEVKPNPLQDANLCSRVFFWWLNPLFKIGHKRRLEEDDMYSVLPEDRSQHLGEELQGFWDKEVLRAENDAQKPSLTRAIIKCYWKSYLVLGIFTLIEESAKVIQPIFLGKIINYFENYDPMDSVALNTAYAYATVLTFCTLILAILHHLYFYHVQCAGMRLRVAMCHMIYRKALRLSNMAMGKTTTGQIVNLLSNDVNKFDQVTVFLHFLWAGPLQAIAVTALLWMEIGISCLAGMAVLIILLPLQSCFGKLFSSLRSKTATFTDARIRTMNEVITGIRIIKMYAWEKSFSNLITNLRKKEISKILRSSCLRGMNLASFFSASKIIVFVTFTTYVLLGSVITASRVFVAVTLYGAVRLTVTLFFPSAIERVSEAIVSIRRIQTFLLLDEISQRNRQLPSDGKKMVHVQDFTAFWDKASETPTLQGLSFTVRPGELLAVVGPVGAGKSSLLSAVLGELAPSHGLVSVHGRIAYVSQQPWVFSGTLRSNILFGKKYEKERYEKVIKACALKKDLQLLEDGDLTVIGDRGTTLSGGQKARVNLARAVYQDADIYLLDDPLSAVDAEVSRHLFELCICQILHEKITILVTHQLQYLKAASQILILKDGKMVQKGTYTEFLKSGIDFGSLLKKDNEESEQPPVPGTPTLRNRTFSESSVWSQQSSRPSLKDGALESQDTENVPVTLSEENRSEGKVGFQAYKNYFRAGAHWIVFIFLILLNTAAQVAYVLQDWWLSYWANKQSMLNVTVNGGGNVTEKLDLNWYLGIYSGLTVATVLFGIARSLLVFYVLVNSSQTLHNKMFESILKAPVLFFDRNPIGRILNRFSKDIGHLDDLLPLTFLDFIQTLLQVVGVVSVAVAVIPWIAIPLVPLGIIFIFLRRYFLETSRDVKRLESTTRSPVFSHLSSSLQGLWTIRAYKAEERCQELFDAHQDLHSEAWFLFLTTSRWFAVRLDAICAMFVIIVAFGSLILAKTLDAGQVGLALSYALTLMGMFQWCVRQSAEVENMMISVERVIEYTDLEKEAPWEYQKRPPPAWPHEGVIIFDNVNFMYSPGGPLVLKHLTALIKSQEKVGIVGRTGAGKSSLISALFRLSEPEGKIWIDKILTTEIGLHDLRKKMSIIPQEPVLFTGTMRKNLDPFNEHTDEELWNALQEVQLKETIEDLPGKMDTELAESGSNFSVGQRQLVCLARAILRKNQILIIDEATANVDPRTDELIQKKIREKFAHCTVLTIAHRLNTIIDSDKIMVLDSGRLKEYDEPYVLLQNKESLFYKMVQQLGKAEAAALTETAKQVYFKRNYPHIGHTDHMVTNTSNGQPSTLTIFETAL BF2D53136B78C0BA abcc4-mpp1-1 8 11 unspecified role unspecified role bait bait purified purified gst tag 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 binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction f1f we investigated if MPP1 bound the C terminus of ABCC4. Purified MPP1 was incubated with the purified C terminus of ABCC4 (100 amino acids) in the absence or presence of various concentrations of an ABCC4 competitor peptide containing or lacking the ABCC4-PDZ motif (Fig. 1f). This in vitro competition experiment demonstrated that the peptide lacking the ABCC4-PDZ motif was incapable of disrupting the MPP1 and ABCC4 protein interaction. Strikingly, the ABCC4-peptide containing the PDZ motif, potently disrupted complex formation with an IC50 of ~300 nM, which suggested a 15-fold greater affinity of MPP1 for ABCC4 vs. glycophorin c. mpp1-abcc4-1 2 11 unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f2b To determine whether endogenous ABCC4 and MPP1 directly interacted in AML cells, we performed co-immunoprecipitation experiments using MO7e cells, a myeloid leukemia cell line derived from a pediatric patient20,21. Reciprocal immunoprecipitation with anti-MPP1 demonstrated that the MPP1 immunoprecipitates contained ABCC4 (Fig. 2b). abcc4-1 2 11 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f2b To determine whether endogenous ABCC4 and MPP1 directly interacted in AML cells, we performed co-immunoprecipitation experiments using MO7e cells, a myeloid leukemia cell line derived from a pediatric patient20,21. Reciprocal immunoprecipitation with anti-MPP1 demonstrated that the MPP1 immunoprecipitates contained ABCC4 (Fig. 2b). abcc4-2 6 11 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range binding region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association f2c To verify that the ABCC4 interaction with MPP1 was mediated exclusively by the C-terminal ABCC4-PDZ motif, cells were co-transfected with an expression vector for MPP1 and either an expression vector for ABCC4 or ABCC4ΔC (lacking the PDZ motif) followed by co-immunoprecipitation with GFP-TRAP. Re-probing the immunoprecipitation with anti-MPP1 antibodies showed that MPP1 formed a protein complex with ABCC4, whereas ABCC4ΔC was incapable of associating with MPP1 (Fig. 2c). abcc4-mpp1-2 7 11 unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role bait bait purified purified gst tag gpires glycosylphosphatidylinositolated residue LIPID GPI-anchor amidated carboxyl end GPIRes GPIanchor glycosylphosphatidylinositol undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding range required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association f2f The MPP1 N terminus, up to the PDZ domain (amino acids 1–83), as well as the SH3 and GUK domain (amino acids 162–466), were incapable of immunoprecipitating ABCC4. The PDZ domain (amino acids 82–160) immunoprecipitated almost identical amounts of ABCC4 as the full-length MPP1, indicating it was necessary and sufficient for ABCC4 binding (Fig. 2f). abcc4-mpp1-3 5 11 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell proximity proximity close-contact colocalization neighbourhood interaction f2g in the AML cell line MO7e, PLA confirmed that endogenous ABCC4 and MPP1 interacted (Fig. 2g). abcc4-mpp1-4 4 11 unspecified role unspecified role neutral component neutral component purified purified binding range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain fluorescin tag fluorescein fluorescein label fluorescein n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction sf1c To confirm that the ABCC4-PDZ motif interacted with MPP1, fluorescence polarization was used to show that a fluorescein-labeled ABCC4-PDZ motif peptide bound to MPP1 has an estimated dissociation constant K D of ~600 nM (Supplementary Fig. 1c). abcc4-mpp1-5 1 11 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell binding range required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell proximity proximity close-contact colocalization neighbourhood interaction sf2 an in cellular proximity ligation assay (PLA) was used to further confirm whether ABCC4 and MPP1 interacted at the plasma membrane due to the PDZ motif. HEK293 cells were transfected with either ABCC4 and MPP1 or ABCC4ΔC and MPP1. After fixation, cells were incubated with anti-ABCC4 and anti-MPP1 antibodies followed by PLA; only cells where ABCC4 and MPP1 interacted produced a fluorescent signal indicating the ABCC4-PDZ motif was required for MPP1 and ABCC4 interaction in cells (Supplementary Fig. 2a). abcc4-mpp1-6 3 11 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range binding range required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell physical association physical association sf3b sf3c We next investigated whether MPP1 affected ABCC4 mobility at the plasma membrane using FRAP (Fluorescence Recovery After Photobleaching). Cells expressing GFP-ABCC4 in the presence or absence of MPP1 were analyzed. MPP1 increased the half-time to fluorescence recovery (τ 0.5) of GFP-ABCC4 by greater than twofold (Supplementary Fig. 3b), whereas co-expression of MPP1 with GFP-ABCC4ΔC did not affect the τ 0.5 (not shown). abcc4-mpp1-7 9 11 unspecified role unspecified role neutral component neutral component purified purified binding range required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain biontin tag biotin tag biotin tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction f5a f5b