Figure 5
From: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
PAX8 is required for RB protein stability. The effect of PAX8 knockdown on RB expression at (a) the transcript and (b) the protein levels. (a) Total RNA was isolated from the indicated sample at 48 h post-transfection, and subjected to qPCR analysis. Gene expression levels were normalized to the expression of the housekeeping genes, PPIB and YWHAZ, and presented relative to the siControl-transfected samples. The data are means±s.d. of three independent experiments. (b) Whole-cell lysates were extracted from siRNA-transfected A498, 786-O, IGROV-1 and K1 cells at 96 h post transfection. The lysates were subjected to immunoblotting using the indicated antibodies. (c) Effects of proteasome inhibition on RB depletion in response to PAX8 knockdown. K1 cells were transfected with the indicated siRNA. After 24 h, cells were transfected either with a vector control (−) or with pCMV-RB (+). At 48 h post DNA transfection, the cells were incubated in medium with or without MG132 for 12 h. Whole-cell lysates were then extracted and subjected to immunoblotting using the indicated antibodies. qPCR, quantitative PCR; RB, retinoblastoma; siRNA, small interfering RNA.
