Figure 6

The role of kinase pathways in VCAM-1 expression in VEC treated without or with insulin or TNFα alone or both and in the absence or presence of kinase inhibitors. Cells were grown to 80% confluence in growth medium. Growth medium was replaced with serum-free medium for 24 h. Cells were pre-treated with either no inhibitor, PD98059 (10 μM), Wortmannin (15 μM), SB203580 (100 nM) or SP600125 (25 μM) for one hour and then treated without or with insulin (10 nM) or TNFα (20 ng/mL) alone, or insulin plus TNFα for 10 additional minutes. Proteins from cleared lysates were analyzed by SDS-PAGE and determined by Western blot analysis for VCAM-1 expression. Representative Western blots of VCAM-1 expression are shown in the absence or presence of indicated inhibitors [Panel A] and without or with insulin (INS) [Panel B] or TNFα [Panel C] alone, or without or with insulin plus TNFα [Panel D]. [Panel E, graph] Changes in amounts of VCAM-1 protein in the absence or presence of inhibitors and without or with insulin or TNFα, or insulin plus TNFα are expressed as percent of controls and are represented as means ± SEM of 5 experiments. *, P < 0.05 vs controls (serum-free medium alone); **, P < 0.05 vs positive controls (insulin or TNFα with no inhibitor). #, P < 0.05 vs insulin plus TNFα (no inhibitors). (SFM) serum-free medium; (INHB) inhibitors; (NI) no inhibitors; (INS) insulin; (TNFα) Tumor Necrosis Factor-alpha; (PD) PD98059; (WT) Wortmannin; (SB) SB203580; and (SP) SP600125.