Fig. 6: OTUB1 participates in the interaction between NY-ESO-1 and PP1α and is required for NY-ESO-1-mediated PP1α deubiquitination.
A Schematic procedure of PP1α interactome analysis by co-IP coupled with LC-MS/MS assay. HEK293T cells were transfected with PP1α-Flag alone or in combination with Myc-NY-ESO-1 and harvested 48 h later. Cell lysates were immunoprecipitated with anti-Flag antibodies, and the final immunoprecipitates were identified using LC-MS/MS. B Venn diagram showing the effect of NY on PP1α interactome. C A table listing the interactive proteins identified by the LC-MS/MS assay along with the corresponding parameters. D Endogenous interaction of NY-ESO-1, OTUB1 and PP1α in A375 cells. E Immunoblot analysis of the interaction between NY-ESO-1, PP1α, and OTUB1 in HEK293T cell lysates following IP with anti-HA mAbs. F Immunoblot analysis of the indicated proteins in HeLa cells transfected with NY-ESO-1 plasmids together with two additional individual siRNAs targeting OTUB1 or control siRNAs. G Immunoblot analysis of the indicated proteins in A375 cells transfected with a mixture of NY-ESO-1 siRNAs (siNY-1:siNY-2, 1:1) along with OTUB1 siRNAs (siOTUB1-1 or siOTUB1-2) and/or NY-ESO-1 plasmids. H, I Immunoblot analysis of PP1α ubiquitination in HeLa cells (H) and A375 cells (I) post indicated transfections. J Schematic of the domain architecture of OTUB1, showing its OTU domain, and ubiquitin-binding domain (UBD), with specific mutations in residues that were used in this study. K Immunoblot analysis of the indicated proteins in HeLa cells transfected with NY-ESO-1 plasmids together with a mixture of OTUB1 siRNAs (siOTUB1-1:siOTUB1-2, 1:1) and wild-type or mutated OTUB1 plasmids. L Immunoblot analysis of PP1α ubiquitination in HeLa cells post indicated transfections. Cells were treated with MG132 for 4–6 h, then lysed with 1%SDS buffer and boiled for 5 min to denature. Subsequently, the lysates were diluted 10-fold with cold IP buffer and subjected to immunoprecipitation using anti-FLAG magnetic beads. P/T PP1α/β-Tubulin, pU/P polyUbK48/PP1α, F/T Flag/β-Tubulin, pU/F polyUbK48/Flag.
