USP3 deubiquitinates KLF5.
A, USP3 decreased KLF5 ubiquitination in a DUB activity–dependent manner. KLF5–3×FLAG and HA-Ub were co-expressed with USP3 or USP3 C168S in HEK293T cells. WB was used to detect the ubiquitinated KLF5 protein levels after the cells were treated with MG132 for 6 h. The experiment was repeated three times, and a representative result is shown. B, USP3 knockdown in HCC1806 and HCC1937 cells increased the endogenous KLF5 protein ubiquitination. The cells were transfected with control or USP3 siRNAs for 48 h. Control siRNA was used as the negative control. Control IgG refers to goat IgG antibody, which is the negative control for KLF5 antibody. WB was used to detect the ubiquitinated KLF5 protein levels after the cells were treated with MG132 for 6 h. The experiment was repeated three times, and a representative result is shown. IP, immunoprecipitation; IB, immunoblotting.