Figure 2.

PCNA regulates the stability of SIVA1. (A) PCNA depletion decreases the stability of SIVA1. HeLa cells were infected with lentiviruses carrying nontarget control or PCNA-specific shRNAs. 48 h later, cell lysates were subjected to immunoblotting using the indicated antibodies. (B) PCNA depletion has no effect on SIVA1 mRNA levels. HeLa cells were infected with lentiviruses carrying nontarget control or PCNA-specific shRNAs. 48 h later, the levels of specific mRNAs were determined by quantitative RT-PCR. Bars represent the mean of three experiments, and error bars are SDs. (C) MG132 treatment partially rescues the levels of SIVA1 protein in PCNA-depleted cells. HeLa cells were infected with lentiviruses carrying nontarget control or PCNA-specific shRNAs. 48 h later, cells were treated with proteasome inhibitor MG132 (10 µM) or DMSO for 6 h. Cell lysates were then subjected to immunoblotting using the indicated antibodies. (D) SIVA1 depletion has no effect on PCNA protein stability. HeLa cells were transfected twice with control siRNA or siRNAs specific for SIVA1. Cell lysates prepared 48 h after the second transfection were analyzed by immunoblotting with the indicated antibodies. (E) RAD18 depletion has no effect on SIVA1 protein stability. HeLa cells transfected with control siRNA or siRNAs specific for RAD18 were treated with 50 J/m² UV for 1 h or left untreated. Cell lysates were then analyzed by immunoblotting with indicated antibodies. ShCon, control shRNA; SiCon, control siRNA; ub, ubiquitin.