Fig. 5. USP4 stabilizes and protects CypA from degradation via deubiquitination.

a Different forced USP4 expression affected the protein levels of CypA, whereas regulated expression of CypA had no effect on USP4 protein levels. b CypA was not regulated by USP4 at the mRNA level. c, d USP4 overexpression increased CypA protein levels endogenously and exogenously in Huh7 cells. USP4 knockdown decreased CypA protein levels endogenously and exogenously in MHCC97H cells. e Impact of USP4 on CypA ubiquitination in vivo. Lysates of 293T cells were immunoprecipitated with an antibody against Flag-tag and analyzed using western blotting analyses to detect poly-ubiquitination of CypA. f The impact of USP4 on CypA ubiquitination in MHCC97H cells. Lysates of MHCC97H cells with USP4-shRNA transfected or not were immunoprecipitated with an antibody against Flag-tag and analyzed using western blotting analyses to detect poly-ubiquitination of CypA. g USP4 enhanced stability of the CypA protein. MHCC97H-pLKO/shUSP4 cells and Huh7-USP4/vector cells were exposed to CHX and collected at the indicated times. Expression of CypA was examined using western blotting assays. The experiments were repeated three times. Western blotting analyses were quantified via densitometry. Error bars represent the SD. **P < 0.01