Fig. 7

Effect of Scribble silencing on Scribble, membrane localization of PHLPP isoforms, and neuronal glucose uptake under insulin sensitive and insulin resistant condition in neuronal cells (N2A). N2A cells were differentiated in serum-free medium in the absence of (MF) or chronic presence of 100 nM insulin (MFI) for 3 days. Three days post-differentiation, membrane and cytosol fraction were isolated and subjected to western blotting, followed by probing with relevant primary antibodies. A Bar represents relative change in Scribble when probed with anti-Scribble antibody. (B-D) Three days post-proliferation, Scribble was silenced using Scribble specific siRNA. N2A cells were differentiated in serum-free medium in the absence of insulin (MF) for 3 days. Three days post-differentiation, membrane and cytosol fraction were isolated and subjected to western blotting, followed by probing with relevant primary antibodies. B Bar represents relative change in Scribble when probed with anti-Scribble antibody. C Bar represents relative change in PHLPP1 probed with anti-PHLPP1 antibody. D Bar represents relative change in PHLPP2 probed with anti-PHLPP2 antibody. E Three days post-proliferation, N2A cells were transfected with Scribble specific siRNA and then differentiated under MF MFI condition for 3 days. Differentiated N2A cells were serum starved for 2 h, followed by 100 nM insulin for 30 min. Uptake of 2-NBDG was then measured. 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. IB Immunoblot; A.U Arbitrary Units