Figure 5
Involvement of the ERK and JNK MAP kinase pathways in CPZ-induced p21Waf1/Cip1 expression. (A) C6 cells were serum-starved with 0.5% serum for 24 h, and then treated with 20 µM CPZ for various lengths of time. Western blotting was performed with total protein extracts using antibodies against phospho-ERK1/2 (Thr 202/Tyr 204), phospho-JNK1/2 (Thr183/Tyr185), or phospho-p38 kinase (Thr180/Tyr182), and then probed with an antibody against total proteins as an internal control. Each blot represents at least three separate experiments. (B) Serum-starved C6 cells were left untreated or treated with U0126, SP600125 (SP), or SB203580 for 30 min, followed by treatment with 20 µM CPZ for an additional 2 h, as indicated. Total cell lysates were prepared and subjected to Western blotting with anti-Egr-1 antibody. The same blot was re-probed with anti-ERK2 antibody as an internal control. Each blot represents at least three separate experiments. (C) C6 cells were transfected with p21-Luc(-150/+38). After 48 h, cells were left untreated or treated with 20 µM CPZ, SP600125 (SP), or U0126, as indicated, for an additional 8 h, and then luciferase activities were measured as in Figure 2C. Error bars represent the mean ± SD of three independent experiments performed in triplicate. (D) Serum-starved C6 cells were left untreated or treated with 10 µM U0126 or 50 µM SP600125 (SP) for 30 min, followed by treatment with 20 µM CPZ for an additional 24 h, as indicated. Total cell lysates were prepared and subjected to Western blotting with anti-p21 antibody. The same blot was re-probed with anti-ERK2 antibody as an internal control. Each blot represents at least three individual experiments.
