Fig. 4.
CHIP-induced OCT4 polyubiquitination at K284 regulates OCT4 stability. a OCT4 ubiquitination at Lys284, with di-glycine modification in the MS/MS spectrum. b MCF7_OCT4KD cells were transfected with the indicated mutant vectors for 40 h, and were treated with 20 μM MG132 for 8 h. Interaction of OCT4 (WT or mutant) with CHIP was detected by performing immunoprecipitation assay. c The cells were transfected with increasing concentrations of the FLAG–CHIP-expressing vector and were cotransfected with OCT4_K284R-expressing vector for 40 h, followed by treatment with 20 μM CHX for 8 h. Mutated OCT4 and FLAG–CHIP levels were determined by performing western blotting, with GAPDH as a loading control. d The cells were transfected with the indicated vectors for 40 h and were treated with 20 μM MG132 for 8 h. Cell lysates were prepared in denaturing condition and were immunoprecipitated using anti-OCT4 antibody. OCT4 polyubiquitination was determined by performing western blotting