FIGURE 7.
O-GlcNAcylation of hepatic 4E-BP1 increases its association with eIF4E. Livers from Ins2Akita/+ diabetic mice and non-diabetic littermates were perfused in situ with a nonrecirculating medium in the absence of insulin. A, phosphorylation of 4E-BP1 was evaluated by Western blot analysis as described in the legend to Fig. 1. Phosphorylation of S6K1 on Thr389 (B) and eIF4G on Ser1108 (C) was measured by Western blot using phosphospecific antibodies. D, O-GlcNAcylation of total 4E-BP1 was measured by Western blot analysis. The association of 4E-BP1 (E) and truncated 4E-BP1 (tr4E-BP1; F) with eIF4E was examined by immunoprecipitating (IP) eIF4E from liver supernatants and measuring the amount of 4E-BP1 in the immunoprecipitate by Western blot analysis. O-GlcNAcylation of full-length 4E-BP1 (G) and tr4E-BP1 (H) bound to eIF4E was measured by Western blot analysis. I, total eIF4E, 4E-BP1, and 4E-BP1 O-GlcNAcylation was assessed in WCL by Western blot analysis. Representative blots are shown. Values are means + S.E. (n = 8). Statistical significance is denoted by *, p < 0.05.