Anti-ICAM-1 stimulation induces an increase in PKCα^{Thr 638} phosphorylation through XO/PLC/ERK1/2 activities but not by oxidation. A,B,D

<p>) 70% confluent monolayers of HMVECLs were pretreated with TNFα to induce ICAM-1 expression. Then, the endothelial cells were were treated for 1 hr with the pharmacological inhibitors allopurinol (0.3 mg/ml), PD98056 (20 µM), U73122 (10 µM), apocynin (4 mM), Ly294002 (100 nM), Go6976 (2.3 nM), PP2 (10 µM), apocynin (4 mM) or the solvent control DMSO (0.01%). The cells were stimulated with anti-ICAM-1-coated beads and examined for (<b>A,B</b>) PKCα^Thr638 phosphorylation and total PKCα by western blot or examined for (<b>D</b>) cytotoxicity by the Vybrant cytotoxicity assay. In panel D, 200 µM H₂O₂ was used as a positive control for cytotoxicity. <b>C</b>) To label non-oxidized cysteines, BIAM was added to the cell lysates. PKCα was immunoprecipitated and BIAM labeling was detected by western blot with HRP-conjugated streptavidin. The blots were reprobed for total PKCα. The positive control for oxidation includes lysates oxidized with 200 µM H₂O₂ for 20 min before addition of BIAM. Loss of BIAM labeling in the western blot indicates PKCα oxidation. Shown are the means ± SEM from 3 experiments. *, p<0.05 compared to nonstimulated (NS) controls.