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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 2: — Effect of PTEN or SHIP2 down-regulation on glucose uptake and insulin signaling upstream of Akt. Scrambled or PTEN- or SHIP2-specific siRNA–transfected N2A cells were differentiated in the absence (MF) or chronic presence (MFI) of insulin for 3 d and stimulated with or without insulin (100 nM) for 30 min. (A) Uptake of 2-DOG was measured in 40-μg cell lysates. Uptake of each sample was measured in duplicate. Bar represents fold change in 2-DOG uptake. (B) The cytosolic fractions were isolated, subjected to Western immunoblotting, and probed with anti-GLUT4 (panel A) or anti-tubulin (panel B) antibodies. Total cell lysates (500 μg) were immunoprecipitated with anti–IR-β (for panels C and D) or anti-IRS1 (for panels E and F) antibodies, subjected to Western immunoblotting, and probed with anti-phosphotyrosine (panels C and E), anti–IR-β (panel D), or anti-IRS1 (panel F) antibodies. For checking knockdown of PTEN and SHIP2, cell lysates were subjected to Western immunoblotting and probed with anti-PTEN (panel G), anti-SHIP2 (panel H), or anti-actin (panel I) antibodies. (C) Bar diagram representing relative densitometric values of GLUT4 expression in cytosolic fractions. (D) Primary cortical neurons were transfected with scrambled or PTEN-specific siRNA, and 2-DOG uptake was measured in 40-μg cell lysates. Uptake of each sample was measured in duplicate. Bar represents fold change in 2-DOG uptake. (E) Bar diagram representing relative densitometric values of pIR-β after normalizing with IR-β expression (pIR-β/IR-β). (F) Bar diagram representing relative densitometric values of pIRS1 after normalizing with IRS1 expression (pIRS1/IRS1). (G) Cell lysates (500 μg) were immunoprecipitated with anti-IRS1 antibody, and PI3K activity was measured. Bar represents change in IRS1-associated PI3K activity. All the experiments were repeated thrice, and a representative result is shown. Values are mean ± SE. **p < 0.01 compared with lane 1; *p < 0.05 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. Open bars, MF; solid bars, MFI. IB, immunoblotted; IP, immunoprecipitated.