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. 2009 Mar 3;58(6):1321–1332. doi: 10.2337/db08-0519

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© 2009 by the American Diabetes Association.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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FIG. 1. — Activation of the mTORC1 pathway by overexpression of FLAG-Rheb in pancreatic β-cells. Islets isolated from mice of each genotype were incubated either in 50% RPMI medium without FCS (A, B, and D) or in RPMI medium (C), or they were stimulated with 100 nmol/l IGF-1 (B) or 15% FCS (D) as indicated. The same amounts of cellular extracts were analyzed by immunoblotting with the antibodies to Rheb, phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein, phospho-4EBP1 (Thr37/46 or Thr70), phospho-p70 S6 kinase (Thr389), p70 S6 kinase, phospho-PKB (Ser473), PKB, phospho-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, or IRS2. A representative experiment is shown. The bottom panel in A shows the same blot as the top panel, except that it was exposed longer to detect the endogenous Rheb (end-Rheb). E: Relative quantification of phospho-S6 (Ser235/236), phospho-4EBP1 (T37/46 or T70), phospho-PKB (Ser473), phospho-p44/42 MAP kinase (Thr202/Tyr204), and IRS2. The immunoblots were scanned, and the optical density for R3 with IGF1 or FCS stimulation (A, B, and D) or the optical density for R3 (C) was set to 100%. F: Immunoprecipitation with the anti-FLAG antibody was performed using lysates of the hypothalamus, muscle, or liver isolated from mice of each genotype and analyzed by immunoblotting with the same antibody. WT, wild type.

FIG. 1. — Activation of the mTORC1 pathway by overexpression of FLAG-Rheb in pancreatic β-cells. Islets isolated from mice of each genotype were incubated either in 50% RPMI medium without FCS (A, B, and D) or in RPMI medium (C), or they were stimulated with 100 nmol/l IGF-1 (B) or 15% FCS (D) as indicated. The same amounts of cellular extracts were analyzed by immunoblotting with the antibodies to Rheb, phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein, phospho-4EBP1 (Thr37/46 or Thr70), phospho-p70 S6 kinase (Thr389), p70 S6 kinase, phospho-PKB (Ser473), PKB, phospho-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, or IRS2. A representative experiment is shown. The bottom panel in A shows the same blot as the top panel, except that it was exposed longer to detect the endogenous Rheb (end-Rheb). E: Relative quantification of phospho-S6 (Ser235/236), phospho-4EBP1 (T37/46 or T70), phospho-PKB (Ser473), phospho-p44/42 MAP kinase (Thr202/Tyr204), and IRS2. The immunoblots were scanned, and the optical density for R3 with IGF1 or FCS stimulation (A, B, and D) or the optical density for R3 (C) was set to 100%. F: Immunoprecipitation with the anti-FLAG antibody was performed using lysates of the hypothalamus, muscle, or liver isolated from mice of each genotype and analyzed by immunoblotting with the same antibody. WT, wild type.