Fig. 4: RBCK1 inhibits the ubiquitination-mediated degradation of RNF31 in HCC cells.
From: RBCK1 promotes hepatocellular carcinoma metastasis and growth by stabilizing RNF31
A Immunofluorescence co-staining of RBCK1 and RNF31 in wild-type PLC/PRF/5 and huh-7 cells to detect colocalization. B Co-immunoprecipitation of endogenous RBCK1 and RNF31 in wild-type PLC/PRF/5 and huh-7 cells. C RNF31 protein level after the knockdown of RBCK1 in PLC/PRF/5 and huh-7 cells was analyzed by western blot. D RNF31 mRNA level after RBCK1 knockdown in PLC/PRF/5 and huh-7 cells was determined by qPCR. E The control and RBCK1 knockdown PLC/PRF/5 and huh-7 cells were treated with CHX (20 μg/mL) at different time points. Whole-cell lysates were prepared and subjected to immunoblotting (left panel), and the RNF31 expression was quantified using Quantity One software (right panel). **p < 0.01. F The control and RBCK1 knockdown PLC/PRF/5 and huh-7 cells were pre-treated with the DMSO, proteasome inhibitor, MG132(10 μM), or the lysosome inhibitor, chloroquine (CQ, 25 μM) for 10 h, and then RBCK1, RNF31, and β-actin were detected via western blot. G The control and RBCK1 knockdown PLC/PRF/5 and huh-7 cells were incubated with MG132 at 10 μM for 10 h. Then cell lysates were prepared and immunoprecipitated with anti-RNF31 antibody and then immunoblotted with the indicated antibodies.
