Fig. 7
ENO2 recruits HSP90 to antagonize CHIP-mediated ubiquitination and degradation of MIF. a Representative liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrum identifying HSP90 as an ENO2-interacting protein, with matched peptide fragments highlighted. Reciprocal Co-IP assays confirming the interaction between endogenous ENO2 and HSP90. HCT-116 (b) and DLD-1 (c) cell lysates were immunoprecipitated with anti-HSP90 or anti-ENO2 antibodies and immunoblotted with the indicated antibodies. IgG served as a negative control. d Co-IP analysis showing that ENO2 enhances the association between HSP90 and MIF. DLD-1 cells were transfected with the indicated plasmids or shRNAs and treated with MG132 prior to immunoprecipitation using an anti-Flag antibody. Immunoblot analysis of MIF protein levels following knockdown (DLD-1, e) or overexpression (HCT-116, f) of ENO2 and/or CHIP, indicating that ENO2 stabilizes MIF in a CHIP-dependent manner. g Ubiquitination assay demonstrating that ENO2 depletion promotes CHIP-mediated ubiquitination of MIF. DLD-1 cells were treated with MG132, followed by immunoprecipitation of MIF and immunoblotting for ubiquitin. h Expression validation of wild-type and lysine-mutant MIF constructs (K32R, K66R, K77R) co-transfected with Flag-CHIP in DLD-1 cells. i Ubiquitination assays identifying lysine residues critical for CHIP-mediated ubiquitination of MIF. DLD-1 cells were co-transfected with HA-tagged ubiquitin, Flag-CHIP, and Myc-tagged wild-type or mutant MIF constructs, treated with MG132, immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-HA antibody
