Figure 3
DCA induces sustained activation of Erk1/2 and p38 but not of JNK. SKGT-4 cells were treated with 300 Ī¼M DCA for indicated times. Treatment with 50 ng/ml PdBu for 30 min (A, B) or 10 Ī¼g/ml anisomycin (ANIS) for 6 hr (C, D, E) were used as positive controls for Erk or p38 and JNK activation, respectively. In panel C the first two lanes correspond to unstimulated and anisomycin stimulated C-6 glioma cells (positive control). Where indicated, cells were incubated in the presence or absence of 50 Ī¼M PD98059 (B, F) or 2 Ī¼M SB203580 (D, F) for 30 min prior to stimulation. Total cellular proteins were standardized to 50 Ī¼g and assessed by Western blotting using antibodies specific for phospho-Erk1/2 and total Erk1/2 (A, B); phospho-p38 and total p38 (C, D); or phospho-JNK and total JNK (E). Total cell lysates were prepared and standardized to 350 Ī¼g. DNA affinity purified proteins were resolved by SDS-PAGE and immunoblotted with anti-Fra-1 or anti-JunB, respectively (F). Unbound proteins were immunoblotted with anti-Ī±-tubulin as loading control. Results are representative of at least three independent experiments.