FIG 5.

p38 MAPK and JNK signaling. Cells maintained in serum and grown to 70% confluence were treated with SKi (10 μM) for 24 h. In certain cases, cells were treated with scrambled, Degs1, SK1, or SK2 siRNA (each at a final concentration of 100 nM) for 48 h or with SB203580 (10 μM) or SP600125 (20 μM) for 30 min prior to treatment with and without SKi. (A) Western blot probed with anti-phospho-p38 MAPK or anti-phospho-JNK antibodies showing the effect of Degs1 siRNA on the phosphorylation levels of p38 MAPK and JNK in response to SKi. (B) Western blot probed with anti-PARP antibody showing the effect of SB203580 (SB) or SP600125 (SP) on PARP cleavage. (C) Bar graph showing the effect of SKi with or without SB203580 on DNA synthesis (*, P < 0.05 for SB203580 versus the control; **, P < 0.05 for SB203580/SKi versus SB203580 alone [n = 3 independent experiments]). (D and E) Western blot probed with anti-phospho-p38 MAPK and anti-phospho-JNK antibodies showing no effect of SK1 siRNA (D) or SK2 siRNA (E) on the phosphorylation levels of p38 MAPK and JNK. (F) Western blot probed with anti-Degs1 antibody showing the effect of SB203580 on Degs1 laddering in response to SKi. Blots were reprobed for GAPDH using anti-GAPDH antibody, anti-p38 MAPK antibody, or anti-JNK antibody to ensure comparable protein loading. Results in panels A, B, and D to F are representative of data from at least 3 independent experiments. Lanes C, control.