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Fig. 8. Preincubation conditions modify S6 kinase and S6 ribosomal protein phosphorylation when cells are tested at the end of preincubation, omitting the 2-h deprivation of FBS and of low-dose insulin. Experiments were carried out exactly as described for Fig. 7, except that the 15-min acute stimulation with 100 nM insulin was administered at the end of the preincubation period (I), and the nonstimulated cells (B) were lysed at the same time, i.e., the 2-h deprivation of FBS and of low-dose insulin was omitted. A and B: representative gel where cells preincubated in 5 mM glucose or in 25 mM glucose + 0.6 nM insulin are analyzed after (I) or without (B) acute stimulation with 100 nM insulin. A was blotted with an antibody to phosphorylated S6 kinase and B with an antibody to phosphorylated S6 ribosomal protein. C: blot for β-tubulin. D–G: representative gels and quantitative analyses of cells preincubated in 5 or 25 mM glucose with or without 0.6 nM insulin, as well as 5 mM glucose and 25 mM glucose + 0.6 nM insulin treated with 100 nM rapamycin for 30 min, analyzed in the basal condition (D and F) and after 15 min stimulation with 100 nM insulin (E and G). Left: blots are analyzed after developing with an antibody against phosphorylated S6 kinase (D and E) or against phosphorylated S6 ribosomal protein (F and G). Right: blots stripped and analyzed with antibodies against total S6 kinase (D and E) or against total S6 ribosomal protein (F and G); n = 7 for cells analyzed without acute insulin stimulation and 11 for cells after stimulation with 100 nM insulin. In A and B: cells with and without acute insulin treatment were analyzed on the same gel. D–G: cells with and without acute insulin stimulation analyzed on separate gels. *P < 0.04 in D and F; P < 0.03 in E, and P < 0.005 and **P < 0.02 in G, all compared with control, preincubated in 5 mM glucose.
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