Fig. 8

Effect of PP1α and PP1γ silencing on AD markers: (A, B and D, E) Proliferated N2a cells were transfected with non-specific (scrambled) and PP1α and PP1γ specific siRNA. Post transfection cells were differentiated in the absence (MF; insulin sensitive) or chronic presence of 100 nM insulin (MFI; insulin resistant) for 3 days. Transfected N2a cells were treated with or without 100 nM insulin for 30 min, lysed and probed with relevant primary antibodies for immunoblotting. Bar represents relative change in A and B pTau (Ser396), C and D BACE. Experiments were executed three times and a representative western blot is shown. Data expressed are mean ± SE. ***p < 0.001 compared to Lane 2, ^θp < 0.05 compared to lane 4, ^δδδp < 0.001 compared to Lane 6. C For thioflavin S staining proliferated N2a cells were transfected with non-specific (scrambled) and PP1α and PP1γ specific siRNA. Post transfection cells were differentiated in the absence (MF; insulin sensitive) or chronic presence of 100 nM insulin (MFI; insulin resistant) for 3 days. Transfected N2a cells were fixed, permeabilized, stained using ThS stain and visualized under an immunofluorescence microscope. Experiments were executed twice and representative images are shown. Scale bar: 100 μm. F and G For Amyloid-β measurement PP1α and PP1γ was silenced and conditioned media was collected, concentrated 2 times using speed vacuum and secreted amyloid-β (1–42) levels were measured using Beta-amyloid (1–42) colorimetric ELISA kit. Experiments were executed twice and average is shown. A and B Tau, D and E GAPDH was used as a loading control. Open bars: MF, filled bars: MFI, IB Immunoblot, SC Scrambled