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. 2011 Apr;25(7):701–716. doi: 10.1101/gad.2002611

Figure 5.

Figure 5.

SKIP is required for Nutlin and TGF-β-induced p21 gene expression. (A, left panel) qRT–PCR analysis of p21 and PUMA mRNA levels. U2OS cells were transfected with control or SKIP siRNA for 48 h, and incubated in the presence or absence of Nutlin (10 μM) for 18 h. (Right panel, lanes 1–4) Protein lysates were subjected to immunoblot analysis. (B) qRT–PCR analysis of p21 mRNA levels. MDA-MB-231 cells were transfected with control or SKIP siRNA for 48 h, followed by incubation in the presence or absence of TGF-β (5 ng/mL) for the indicated times. (C, lanes 1–6) Immunoblot analysis of SKIP, Smad2/3, p53, p21, or GAPDH in cells transfected with control or SKIP siRNA for 48 h, followed by treatment with TGF-β (5 ng/mL) for the indicated times. (D, left) qRT–PCR analysis of p21 mRNA levels in HCT116 p53−/− cells, H1299 cells, HeLa cells and MDA-MB-231 cells transfected with control or SKIP siRNA for 48 h. (Right, lanes 1–8) Protein lysates were subjected to immunoblot analysis. All of the mRNA expression levels were normalized to GAPDH mRNA, and are represented as fold increase or decrease over untreated cells. Error bars represent the standard deviation obtained from three independent experiments.