Figure 4

RT-PCR (each left panel ) and Western blot (each right panel ) results. (A) HEK293T cells were transfected with a vector encoding flag-SPOP and SPOP-specific shRNA vectors (shSPOP1 and shSPOP2) or a negative control vector (shControl). After incubation for 48 h, total RNAs were prepared and subjected to reverse transcription-PCR for SPOP and actin mRNAs. Cell lysates were subjected to western blot with anti-FLAG antibody. (B) HEK293T cells were transfected with p53-specific shRNA/miRNA vectors (shP53/mirP53) or a negative control vector (shControl/mirControl). Anti-p53 antibody was used. (C) demonstrates the RT-PCR and western blot results for EGFP gene silencing. (D) and (E) demonstrate the RT-PCR and western blot results for firefly luciferase gene silencing.