Fig. 6

Effect of PP1α and PP1γ silencing on MLK3 and IKK in insulin sensitive and insulin resistant condition: A–D 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 pIKKα/β (Ser179/180) C and D pMLK3 (Ser674). Experiments were executed three times and a representative western blot is shown. Data expressed are mean ± SE. **p < 0.01, compared to Lane 1, ^###p < 0.001 compared to Lane 2, ^θθθp < 0.001 compared to lane 4. A and B IKKα, C and D MLK3 was used as a loading control. Open bars: MF, filled bars: MFI, IB Immunoblot, SC Scrambled. (E) (i) Under basal condition, when PP1γ is present and insulin is not present, MLK3 is phosphorylated at its inhibitory site leading to its inactivation. Inactivated MLK3 cannot phosphorylate and activate IKK which in turn leads to the activation of GSK3α. (ii) Under insulin stimulated condition, when PP1γ is present it removes inhibitory phosphorylation of MLK3 and activating it. Activated MLK3 phosphorylates and activates IKK causing GSK3α phosphorylation and inactivation. (iii) When PP1γ was downregulated, it cannot dephosphorylate MLK3 causing its inactivation. Inactivated MLK3 in turn cannot phosphorylate and activate IKK leading to activation of GSK3α. Created with BioRender.com