Fig. 4

(A) Dose-response effects of gp120 on tubular cell p38 MAPK phosphorylation/activation. Equal numbers of tubular cells were incubated in media containing gp120 (1, 10, and 100 ng/ml) for 1 hr. Subsequently; cells were lysed and processed for p38 MAPK activity. P38 MAPK activity in cultured human proximal tubular cells was measured by an in vitro immunokinase assay using ATF-2 as the substrate for the reaction. The upper panel shows the effect of gp120 on the activation of p38 MAPK. Middle panel: Western blot using specific phospho-antibody to p38 MAPK. Equal numbers of tubular cells were incubated in media containing variable concentrations of gp120 (0–100 ng/ml) for 1 hr. At the end of the incubation period, cells were lysed, immunoprecipitated with phospho-p38 MAPK antibody, and probed for p38 MAPK. The middle panel shows the effect of gp120 on tubular cell p38 MAPK phosphorylation. The lower panel shows total p38 MAPK activity. The line diagram shows the cumulative data of three experiments. (B) Time course effects of gp120 on phosphorylation of tubular cell activation of p38 MAPK. P38 MAPK activity in human proximal tubular cells was measured by an in vitro immunokinase assay using ATF-2 as the substrate for the reaction. Equal numbers of tubular cells were incubated in media containing gp120 (10 ng/ml) for indicated time periods. Subsequently, cells were lysed and processed for p38 MAPK activity. The upper panel shows the effect of gp120 on the activation of tubular cell p38 MAPK. Middle panel: Western blot using specific phospho-antibody to p38 MAPK. Equal numbers of tubular cells were incubated in media containing gp120 (10 ng/ml) for indicated time periods. At the end of the incubation periods, cells were lysed, immunoprecipitated with phospho-p38 MAPK antibody, and probed for p38 MAPK. The middle panel shows the effect of gp120 on tubular cell p38 MAPK phosphorylation. The lower panel demonstrates total p38 MAPK activity. The line diagram shows the cumulative data of three experiments. (C) Effect of SB 202190 on tubular cell p38 MAPK activity (by in vitro immunokinase assay using ATF-2 as a substrate for the reaction). Equal numbers of tubular cells were incubated in media containing either buffer (control; lane 1); gp120 (10 and 100 ng/ml; lanes 2 and 3 respectively); gp120, 100 ng/ml + SB 202190, 1 µM (lane 4); gp120, 10 ng/ml + SB 202190, 1 µM (lane 5); gp120, 100 ng/ml + SB 202190, 5 µM (lane 6); gp120, 10 ng/ml + SB 202190, 5 µM (lane 7); gp120, 100 ng/ml + SB 202190, 10 µM (lane 8); or gp120, 10 ng/ml + SB 202190, 10 µM (lane 9).