P38 mediates the increase in [Ca
to induce NADPH oxidase-derived ROS generation. (A) Activation of P38 was analyzed in Khz-cp-treated SNU-1 cells by immunoblot analysis of phosphorylated P38. (B) Cells were pretreated with 20 μM SB203580 (SB) and SB202474 (SBA) for 1 h. Apoptosis was analyzed 1 h after Khz-cp treatment as in Figure 2(C). (C) SNU-1 cells were pretreated with P38 inhibitors as in (B), and cytochrome c levels in the cytosol were analyzed by immunoblotting 2 h after Khz-cp treatment. (D, E) SNU-1 cells were transfected with control or P38-1 siRNA. After 48 h, ROS generation was analyzed 30 min after Khz-cp treatment using the Amplex Red hydrogen peroxide assay. The data represent the mean ± SD values (D). P38 protein expression was analyzed by immunoblotting 48 h after transfection (E). (F, G) SNU-1 cells were pretreated with 20 μM SB203580 (SB) and SB202474 (SBA) for 1 h, and ROS generation induced by Khz-cp treatment was measured using the Amplex Red hydrogen peroxide assay. The data represent the mean ± SD values (F). The levels of p47phox and p67phox proteins in the membrane fractions of SNU-1 cells were analyzed by immunoblotting 15 min after Khz-cp treatment (G).