Fig. 6

ANXA1 Recruits Deubiquitinating Enzyme USP5 to Deubiquitinate and Stabilize GOT1. A-C: Venn diagram based on IP-MS (ANXA1 and GOT1) with Molecular Signatures Database(MsigDB) to find deubiquitinating enzymes that stabilize GOT1 and IP-MS analysis as well as secondary mass spectrometry diagrams of USP5 and GOT1. D: Multiplex immunofluorescence detecting the co-localization of ANXA1, USP5, and GOT1 in ICC tissues. E: CO-IP detecting the interaction between ANXA1 and USP5 in HUCCT1 cells endogenously. F: CO-IP detecting the interaction between ANXA1 and USP5 in 293T cells exogenously. G: IB of GOT1, ANXA1, and α-tubulin in HUCCT1 cells transduced with sh-USP5 or sh-control after CHX treatment (100 µg/ml)for the indicated times (left). A graph showing normalized GOT1 levels is also shown (right). (Mean values (n = 3) ± s.d. two-way ANOVA, **p < 0.01.). H: IB of GOT1, USP5, and α-Tubulin in HUCCT1 and HCCC9810 cells transduced with sh-control or sh-USP5 after treatment with MG132 (10 μm, 6 h). I: IP with anti-Flag antibody and IB of HA-Ub, Flag-GOT1, USP5, and α-Tubulin in HUCCT1 and HCCC9810 cells transfected with the indicated plasmids after treatment with MG132 (10 μm, 6 h). J: IP (using anti-USP5 or anti-GOT1 antibody) and IB of GOT1, ANXA1, and USP5 in HUCCT1 and HCCC9810 cells transduced with sh-control or sh-ANXA1 after treatment with MG132 (10 μm, 6 h). K:IB of GOT1, USP5, and α-Tubulin in HUCCT1 and HCCC9810 cells transduced with sh-control or sh-USP5 and with ANXA1 knockdown. L: IB of GOT1, ANXA1, and α-Tubulin in RBE cells transfected with vector or Flag-ANXA1 and with USP5 knockdown