Figure 8

Effects of kinase inhibitors on insulin augmented TNFα-stimulated NFκB nuclear import. VEC were cultured in growth medium until 80% confluent and then cultured in serum-free medium for 24 h. Thereafter, cells were pre-treated with no inhibitor, PD98059 (10 μM), Wortmannin (15 μM), SB203580 (100 nM) or SP600125 (25 μM) for one hour and then treated with no analog (NA)(i.e., no insulin or TNFα) or with insulin (10 nM), TNFα (20 ng/mL) alone or in combination for 60 minutes. Representative Western blots at time 60 minutes are shown noting changes in relative NFκB protein content in cytoplasmic (C) and nuclear (N) fractions of cells treated with designated inhibitors alone [Panel A] or without or with inhibitors and in the absence or presence of insulin plus TNFα [Panel B]. [Panel C] Percentage of nuclear NFκB is expressed for cells treated with either no analog (NA) (open bars) or presence of insulin (striped bars) or TNFα (shaded bars) alone, or insulin plus TNFα (solid bars) and in the absence or presence of indicated inhibitor. Graph represents the mean ± SEM for 5 separate experiments. (NA) no analog; (NO INHB) no inhibitor; (INS) insulin; (TNFα) Tumor necrosis factor-alpha; (INS + TNFα) insulin plus TNFα; (PD) PD98059; (WT) Wortmannin; (SB) SB203580; (SP) SP600125. *, P < 0.05 vs controls (no analog no inhibitor; serum-free medium alone); **, P < 0.05 vs TNFα plus WT.