Fig. 5
ENO2 Stabilizes MIF by Inhibiting Ubiquitin-Mediated Degradation. a Proximity ligation assay (PLA) demonstrating the interaction between endogenous ENO2 and MIF in cells. Nuclei were stained with DAPI. b Western blot analysis of MIF protein levels upon knockdown of ENO2 (left, DLD-1 cells) or overexpression of ENO2 (right, HCT-116 cells) compared to non-targeting controls. c Western blot analysis of MIF protein levels in HCT-116 cells overexpressing ENO2 or DLD-1 cells with ENO2 knockdown; both were treated with the proteasome inhibitor MG132. d Western blot analysis of MIF protein levels under various conditions: knockdown of ENO2 (left, DLD-1 cells), knockdown of CHIP, or overexpression of ENO2 (right, HCT-116 cells) and CHIP as indicated. The corresponding expression levels of ENO2, CHIP, and MIF are shown. e Western blot analysis assessing the polyubiquitination level of wild-type or a putative mutant Flag-tagged ENO2 protein in cells treated with MG132. f MIF protein levels in cells expressing wild-type or mutant Flag-tagged ENO2. g Western blot analysis of MIF downstream signaling pathway proteins in ENO2-modulated cancer cells. The phosphorylation levels of STAT3 (p-STAT3) and P65 (p-P65), along with their total protein levels, were examined. β-Actin served as a loading control
