Fig. 3.

CHIP E3 ligase interacts with OCT4 and induces its polyubiquitination. a Interaction between CHIP and OCT4 in MDA-MB-231 cells was detected by performing immunoprecipitation assay. b HEK293 cells were transfected with vectors expressing WT FLAG–CHIP or the indicated CHIP mutants K30A and H260Q and were cotransfected with OCT4 expression vector for 40 h and were treated with 20 μM MG132 for 8 h. Interaction of OCT4 with CHIP (WT or mutant) was detected by performing immunoprecipitation assay. c The cells were transfected with the indicated vectors for 40 h and were treated with 20 μM CHX for 8 h. GAPDH was used as a loading control. MDA-MB-231 cells were transfected with CHIP siRNA (d), FLAG–CHIP-overexpressing vector (e), and WT or mutant FLAG–CHIP-expressing vector (f). After 40 h, the transfected cells 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 detected by performing western blotting