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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 3. — Effect of PTEN or SHIP2 down-regulation on MAPK activation. 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) Cell lysates were subjected to Western immunoblotting and probed with anti–phospho-ERK (panel A), anti-ERK (panel B), anti–phospho-p38 (panel C), anti-p38 (panel D), anti–phospho-JNK (panel E), or anti-JNK (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. (B) Bar diagram representing relative densitometric values of pERK after normalizing with ERK expression (pERK/ERK). (B) Bar diagram representing relative densitometric values of pp38/pJNK after normalizing with p38/JNK expression (pMAPK/MAPK). 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. Open bars, MF; solid bars, MFI. IB, immunoblotted.