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. 2012 Oct 1;23(19):3882–3898. doi: 10.1091/mbc.E12-05-0337

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© 2012 Gupta and Dey. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Effect of ERK inhibition on PTEN-down-regulation–mediated impaired insulin signaling and glucose uptake. N2A cells were differentiated in the absence (MF) or chronic presence (MFI) of insulin for 3 d and pretreated with vehicle (DMSO) or U0126 (25 μM) for 1 h before stimulation with or without insulin (100 nM) for 30 min. (A) Cell lysates were subjected to Western immunoblotting and probed with anti–phospho-ERK (panel A), anti-ERK antibody (panel B), anti–phospho-p38 (panel C), anti-p38 (panel D), anti–phospho-JNK (panel E), or anti-JNK (panel F) antibodies. (B) Uptake of 2-DOG was measured in the presence of vehicle or U0126 (25 μM). Uptake of each sample was measured in 40-μg cell lysates in duplicate. Bar represents fold change in 2-DOG uptake. (C) Cell lysates (500 μg) were immunoprecipitated with anti-IRS1 antibody, subjected to Western immunoblotting, and probed with anti-phosphotyrosine or anti-IRS1 antibodies. Bar represents relative densitometric values of pIRS1 after normalizing with IRS1 expression (pIRS1/IRS1). (D) Cell lysates (500 μg) were immunoprecipitated with anti-IRS1 antibody, and PI3K activity was measured. Bar represents fold change in IRS1-associated PI3K activity. (E) Cell lysates were subjected to Western immunoblotting and probed with anti–phospho-Akt (Ser-473; panel A), anti-Akt (panel B), anti–phospho-GSK3β (Ser-9; panel C), or anti-GSK3β (panel D) antibodies. (F) Bar diagram representing relative densitometric values of pAkt (Ser-473) after normalizing with Akt expression (pAkt/Akt). (G) Bar diagram representing relative densitometric values of pGSK3β (Ser-9) after normalizing with GSK3β expression (pGSK3β/GSK3β). All experiments were repeated thrice, and a representative result is shown. Values are mean ± SE. **p < 0.01 compared with lane 1; ^$$p < 0.01 compared with lane 2; ^##p < 0.01 compared with lane 3; ^θθp < 0.01 compared with lane 4, ^≠≠p < 0.01 compared with lane 9; ^γγp < 0.01 compared with lane 10; ^μμp < 0.01 compared with lane 11; ^∆∆p < 0.01 compared with lane 12. Open bars, MF; solid bars, MFI. IB, immunoblotted; IP, immunoprecipitated.