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. 2005 Nov 22;392(Pt 2):345–352. doi: 10.1042/BJ20051194

Figure 5. Induction of IRS1 protein by insulin is not mediated by proteasomal inhibition or translational activation.

Figure 5

(A) Starved H4IIE cells (SF) were pre-incubated with or without 10 μM MG132 (proteasome inhibitor) for 4 h prior to a 1 h incubation in the absence or presence of 10 nM insulin (Ins) and Western blot analysis of IRS1 and β-actin expression. (B) Mdm2 expression was assessed in the samples from (A). (C) After overnight serum deprivation, H4IIE cells were pre-incubated with or without 10 μg/ml cycloheximide (CHX) or puromycin (PUR) for 30 min, and then treated with 10 nM insulin±each drug for 1 h prior to Western blot analysis and quantification. (D) Phosphorylation of ribosomal S6 protein (P-S6) was determined by Western blot analysis of protein lysates from starved, cycloheximide (CHX; 10 μg/ml)- or insulin (10 nM)-treated cells (1 h incubations). For (A) and (C), values are the ratio of IRS1 to β-actin, and are the average±S.E.M. of at least two experiments performed in duplicate. ***P<0.001; **P<0.01 (experimental compared with serum-free); NS, not significant. For (B) and (D), representative blots from two experiments are shown.