Fig. 2

Depletion of RB1 mRNA and protein expression level caused by gene knock-out in R552X cells. (A) Schematic of performed qRT-PCR using 17 primer sets, black rectangles, for amplifying exon junctions on RB1 cDNA synthesized from RB1 mutated mRNA in R552X cells. Nine independent measurements analyzed for each primer set. (B) Overall RB1 mRNA expression level of WT, He-R552X, and Ho-R552X cell lines measured by qPCR using the 17 primer sets. The average expression level of WT cells was normalized to 1, and the expression levels of He- and Ho-R552X were adjusted accordingly (mean ± s.e.m.). (C) Representative images of western blot analysis using four anti-pRb antibodies recognizing different epitopes. (D) Protein expression level of pRb measured by three or four independent western blot analysis (n = 3 or 4; mean ± s.e.m.). Four different antibodies used to recognize spatially separated epitopes. The average expression level of WT cells was normalized to 1, and the expression levels of He- and Ho-R552X were adjusted accordingly. P values were obtained using one-way analysis of variance (ANOVA) and Tukey’s test for post hoc analysis: *P < 0.05, **P < 0.01, ***P < 0.001. (E) Overall pRb expression level of WT, He-R552X, and Ho-R552X cells. The average expression level of WT cells was normalized to 1, and the expression levels of He- and Ho-R552X were adjusted accordingly (mean ± s.e.m.).