FIGURE 6:

Role of PTEN in FAK-mediated negative regulation of neuronal insulin signaling. (A) Scrambled or PTEN-specific siRNA–transfected N2A cells were differentiated in the absence (MF) or chronic presence (MFI) of insulin. Cell lysates were subjected to Western immunoblotting and probed with anti-phosphotyrosine or anti-FAK antibodies. Bar represents relative densitometric values of pFAK after normalizing with FAK expression (pFAK/FAK). Open bars, MF; solid bars, MFI. (B) Scrambled or FAK-specific siRNA–transfected N2A cells were differentiated in the chronic presence of insulin (MFI) for 3 d and stimulated with or without insulin (100 nM) for 30 min. Cell lysates were subjected to Western immunoblotting and probed with anti–phospho-ERK (panel A), anti-ERK (panel B), anti-FAK (panel C), or anti-tubulin (panel D) antibodies. Bar represents relative densitometric values of pERK after normalizing with ERK expression (pERK/ERK). (C) N2A cells were differentiated in the absence (MF) or chronic presence (MFI) of insulin for 3 d and pretreated with vehicle (DMSO) or wortmannin (1 μM) for 30 min before stimulation with insulin (100 nM) for 30 min. Cell lysates (500 μg) were immunoprecipitated with anti-FAK antibody, subjected to Western immunoblotting, and probed with anti–phosphotyrosine or anti-FAK antibodies. (D) 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. Cell lysates were subjected to Western immunoblotting and probed with anti–phospho-PTEN (Ser-380/Thr-382/383) or anti-PTEN antibodies. Bar represents relative densitometric values of phospho-PTEN after normalizing with PTEN expression (pPTEN/PTEN). All experiments were repeated thrice, and a representative result is shown. Values are mean ± SE. **p < 0.01 compared with lane 1; ^$p < 0.01 compared with lane 3; ^$p < 0.05 compared with lane 2. IB, immunoblotted; IP, immunoprecipitated.