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. 2008 Jul 10;118(8):2959–2968. doi: 10.1172/JCI34277

Figure 8. Hypothalamic overexpression of DN-S6K or Raptor/ΔCT and functional validation of these viruses in hypothalamic GT1-7 cells and rat MBH.

Figure 8

(A) Construction of kinase-dead DN-S6K. (B) Confirmation of DN-S6K expression in the MBH. (C) Construction of Raptor/ΔCT. A part of the hinge domain and all WD repeat domains were removed. The N terminus had Myc and Flag tags. (D) Confirmation of Raptor/ΔCT expression in the MBH. MBH lysate was immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Myc antibody. (E) Adenovirus-infected GT1-7 cells were stimulated with 0, 1, 10, or 100 nM insulin for 30 min. Serine phosphorylations in the insulin-stimulated state of S6 and IRS-1 at Ser636/639 and Ser307 were significantly reduced by DN-S6K and Raptor/ΔCT overexpression (phosphoproteins/total protein ratio, P < 0.01, 2-way repeated-measures ANOVA; n = 2). Representative bands are shown. (F) Overexpression of DN-S6K suppressed phosphorylation of S6 in the MBH of rats fed HFD for 1 d. (G) Overexpression of Raptor/ΔCT suppressed S6K activity in the MBH of 1 d HFD-fed rats. *P < 0.05 versus LacZ.