FIG. 9.

Reversibility of the effect of IL-6 on IRS-1, JNK, and ERK1/2 phosphorylation in C57BL/6J mouse aortas. Three-month-old C57BL/6J female mice were injected i.p. with rhIL-6 1 h before insulin stimulation. In experiments using JNK inhibitor I or PD98059, these were injected i.p. at 10 mg/kg of body weight 2 h before IL-6 injection. Animals were then stimulated with insulin (i.p., 0.75 mU/g of body weight). The aortas were immediately collected and snap-frozen in liquid nitrogen. Equal amounts of aortic lysates were subjected to Western blotting with the appropriate specific phosphoantibodies as described in Materials and Methods. To normalize the blots for protein levels, blots were stripped and reprobed with primary antibodies against the total unphosphorylated form of the indicated protein. (a) ERK1/2 phosphorylation (lower blot) and total levels of ERK1/2 (upper blot). (b) JNK phosphorylation (lower blot) and total levels of JNK (upper blot). (c) Ser⁶¹² phosphorylation of IRS-1(lower blot) and total levels of IRS-1 (upper blot). IP: immunoprecipitation. (d) Ser³⁰⁷ phosphorylation of IRS-1(lower blot) and total levels of IRS-1 (upper blot). Each bar represents the mean ± SD of five independent experiments; the blots shown are from representative experiments. WB: Western blot. *: levels in aortas of IL-6-treated mice versus levels in aortas of control mice; P < 0.01. #: levels in aortas of IL-6-treated mice after injection of either JNK inhibitor I or PD98059 or both versus levels in aortas of IL-6-treated mice; P < 0.01. +: present. −: absent.