RT-qPCR analysis. 293T cells were infected with CUL5-specific shRNA lentiviral particles, total RNA was extracted from wild-type and knockdown cells, RT-qPCR was performed using gene-specific primers. ∆Ct (CtKD-CtWT) was used to calculate mRNA reduction (%) between wild-type and knockdown cells using the following formula: (1-1/2∆Ct) x 100%.

Western blotting analysis.CUL5 protein expression in wild-type (WT) and shRNA knockdown (KD) 293T cells was detected using Western blotting.  Hsp90 α served as a loading control. The blots were incubated with primary antibodies against CUL5 and Hsp90 α, respectively, followed by incubating with HRP-conjugated goat anti-rabbit secondary antibody. Images were developed using NaQ™ ECL Substrate Kit.