Fig. 1
Proteomic analysis of FACI-interacting proteins by BioID and IP-MS. A Schematic diagram of the BioID workflow. Myc-BioID2-FACI fusion protein was used as the bait. B AML12 cell lines stably expressing myc-BioID2-FACI (AML12-BioD2-FACI) and myc-BioID2 (AML12-BioD2) were generated and verified by immunoblotting. C Immunoprecipitation (IP). Biotin-labeled proteins from AML12-BioID2 and AML12-BioID2-FACI cells were captured by streptavidin and separated by SDS-PAGE. Samples were probed with anti-FACI (left) and HRP-streptavidin (HRP-Strep; right). D Schematic diagram of the IP-MS workflow. V5-tagged FACI protein was used as the bait. E AML12 cell lines stably expressing V5-FACI (AML12-V5-FACI) were generated and verified by immunoblotting. A mock AML12 stable cell line (AML12-CTR) was generated and used as a control. F Immunoprecipitation (IP). Protein lysates from AML12-CTR and AML12-V5-FACI cells were incubated with anti-V5 antibody. Immunoprecipitated proteins were separated by SDS-PAGE, followed by immunoblotting (left) and silver staining (right). G Volcano plots illustrating two independent BioID assays (BioID-R1 and -R2) and two independent IP-MS assays (IP-R1 and -R2) in AML12 cells. Y-axis represents log2 fold change (FC) of protein iBAQ intensity of the FACI-immunoprecipitated group relative to the control group. X-axis indicates protein iBAQ values of the FACI-immunoprecipitated group. FACI-immunoprecipitated group refers to AML12-V5-FACI or AML12-BioID2-FACI. Control group refers to AML12-CTR or AML12-BioID2. Proteins with iBAQ value larger than 21.5-fold, protein score higher than 30 and MSMS counts of greater than 2 relative to the control were recognized as “potential FACI interactors” and highlighted in red. The detailed information of “potential FACI interactors” was listed in Additional file 2: Table S2 and Additional file 3: Table S3. iBAQ: intensity-based absolute quantification. H STRING protein–protein interaction networks of FACI-interacting proteins. The 32 high-confidence FACI-interacting proteins were selected from the potential FACI-interactors which were identified more than twice in BioID and IP-MS assays. Network nodes were differentially colored based on protein functions. Detailed information of the 32 FACI-interacting proteins were listed in Additional file 4: Table S4. I The bubble plot depicting gene ontology (GO) of FACI-interacting proteins. Y-axis represents the GO terms. X-axis indicates gene ratio. Bubble colors represent -log10(FDR) and bubble sizes indicate the number of genes. J Schematic diagram of “potential FACI interactors” involved in CCPs