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. 2016 Aug 20;7(41):66740–66753. doi: 10.18632/oncotarget.11458

Figure 8. Y-27632 alters phosphorylation of p53 and expression of p53 target genes.

Figure 8

Y-27632-treated cells retain p53, p16 and phospho-RB protein levels as assessed by immunoblot analysis. Relative protein levels in cells were determined by immunoblot analysis, which showed that immortalized HFK/Myc express similar levels of p16, p53 and phospho-and total pRB protein (A). Y-27632 did not alter level and localization of p53 (B), HPV E6 expressing was used as control for p53 degradation. Interestingly, p21, which is p53 target protein, was decreased (C), which suggest that there is some p53 and pRB independent mechanism of cell cycle regulation. The following antibodies were used to detect the corresponding proteins: p53 (Cell Signal; 1:1,000), pRB (Cell Signal; 1:1,000), and P-pRB (Cell Sign 1:1,000), p16 (Santa Cruz, 1:1,000) and p21 (Santa Cruz, 1:1,000) Secondary antibodies were goat anti-rabbit antibody conjugated to alkaline phosphatase for p53 and pRB or goat anti-mouse antibody for p21, and beta-actin. Beta actin was used as a loading control (Sigma; 1:10,000).