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. 2000 Sep;11(9):2915–2932. doi: 10.1091/mbc.11.9.2915

Figure 2.

Figure 2

Prolonged activation of the MAPK cascade results in increased expression of the cyclin dependent kinase inhibitor p21. (A) Wild-type hepatocytes. (B) p21 null hepatocytes. (C) Wild-type hepatocytes with p21 antisense mRNA. (D) Hep G2 cells. (E) Hep G2 cells with p21 antisense mRNA. Hepatocytes or HepG2 hepatoma cells were infected with kinase active ΔB-Raf: ER poly-L-lysine adenovirus (250 m.o.i), followed by culture as in Methods. In some experiments and in addition to ΔB-Raf:ER infection, cells were also infected with either a null recombinant adenovirus or a p21 antisense mRNA recombinant adenovirus (at 100 m.o.i. each). After 24 h to allow protein expression, cells were treated with either vehicle control or with 100 nM 4-hydroxytamoxifen for 36 h (total time in culture 60 h). Protein expression of p21 was determined by immunoblotting. Equal protein loading (200 μg) per lane; exposures of x-ray film for the ECL immunoblots shown were from 30 s (Panel A) to 5 min (Panels B and C). Lane 1: ΔB-Raf:ER + vehicle control. Lane 2: ΔB-Raf:ER + 4-hydroxytamoxifen. Lane 3: ΔB-Raf:ER + 4-hydroxytamoxifen + 50 μM PD98059. Lane 4: vector control + 50 μM PD98059 + 4-hydroxytamoxifen. A representative experiment for each cell type/condition is shown (n = 6).