FIG. 7.

Effects of stealth siRNA ERK1/2 or JNK1/2 on insulin-stimulated IRS-1 phosphorylation in HUVECs. HUVECs were transfected according to the manufacturer's protocol in antibiotic-free and serum-free Dulbecco's modified Eagle medium with Lipofectamine 2000 (3.5 μl/ml) and each stealth siRNA (100 nM) or the pooled Erk1-Erk2 siRNAs (200 nM) and JNK1-JNK2 siRNAs (200 nM); a scrambled siRNA was used as a negative control. The cells were then incubated for 30 min in the presence or absence of 20 ng/ml of IL-6. Cell lysates were subjected to Western blot analysis as indicated in Materials and Methods. Each bar represents the mean ± SD of four independent experiments; the blots shown are from representative experiments. (a) Ser⁶¹⁶ phosphorylation of IRS-1 (lower blot) and total levels of IRS-1 (upper blot); (b) Ser³¹² phosphorylation of IRS-1 (lower blot) and total levels of IRS-1 (upper blot). IP, immunoprecipitation; WB, Western blot. *: levels in IL-6-treated versus levels in non-IL-6-treated cells; P < 0.01. #: levels in insulin-stimulated, IL-6-treated, specific-siRNA-transfected cells versus levels in insulin-stimulated, IL-6-treated cells transfected with a scrambled siRNA; P < 0.01. +: present. −: absent.