Fig. 5

Effect of PHLPP2 over-expression on PHLPP isoforms, Akt isoforms and AS160 in insulin signaling and insulin resistant condition in neuronal cells (N2A). Three days post-proliferation, PHLPP2 was over-expressed using PHLPP2 specific plasmid. N2A cells were differentiated in serum-free medium in the absence of (MF) or chronic presence of 100 nM insulin (MFI) for 3 days. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. A Bar represents relative change in PHLPP2 probed with anti-PHLPP2 antibody. B Bar represents relative change in PHLPP1 probed with anti-PHLPP1 antibody. C Bar represents relative change in pAkt1 (Ser-473) probed with anti-Akt1 antibody. D Bar represents relative change in pAkt2 (Ser-474) probed with anti-Akt2 antibody. E Post-insulin stimulation, lysates were subjected to immunoprecipitation using anti-Akt3 antibody. Bar represents relative change in pAkt (Ser-473) probed with anti-Akt3 antibody. F Bar represents relative change in pAS160 (Ser-588) when probed with anti-AS160 antibody. G Bar represents relative change in pAS160 (Thr-642) when probed with anti-AS160 antibody. GAPDH has been used as a loading control. Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 compared to lane 1, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 as compared to lane 2, ^$$$P < 0.001, ^$$P < 0.01 as compared to lane 6. IB Immunoblot; IP Immunoprecipitation