FIGURE 8.

Phosphorylation of MAPK kinases 3/6 and of p38 MAPK induced in INS-1-OE cells by thapsigargin is suppressed by the iPLA₂β inhibitor BEL and by the Rho family small G-protein inhibitor C. difficile toxin B, respectively. In A, stably transfected INS-1 cells that overexpress iPLA₂β were preincubated (1 h, 37 °C) with BEL (10 μm) or DMSO vehicle alone. Then the medium was replaced by fresh medium containing 1 μm or no thapsigargin without or with BEL (10 μm), and the cells were again incubated (30 min, 37 °C). At the end of that incubation interval, cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting with antibody directed at phosphorylated MAPK kinases 3/6 (MKK3/6) or against β-actin. Mean values (n = 3) are displayed for the densitometric ratios of the bands for phospho-MKK3/6 and β-actin, and S.E. values (error bars) are indicated. *, p < 0.05 for the value at the indicated condition versus the condition with 1 μm thapsigargin and 10 μm BEL. In B, stably transfected INS-1 cells that overexpress iPLA₂β were preincubated (1 h, 37 °C) with C. difficile toxin B (2 ng/ml) or blank diluent alone. Then the medium was replaced by fresh medium containing 1 μm or no thapsigargin, and the cells were again incubated (30 min, 37 °C). At the end of that incubation interval, cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting with antibody directed at phosphorylated p38 MAPK or total p38 MAPK, as in Fig. 1. The densitometric ratios of the bands for phospho-p38 and total p38 were then determined. Mean values (n = 3) are displayed, and S.E. values are indicated. *, p < 0.05 for the value at the indicated condition versus the condition with 1 μm thapsigargin and 2 ng/ml toxin B.