Fig. 4: Overexpression of IKKα inhibits phosphatase activity of PP1α.

a–d Human NSCLC HCC827 (a, c) and H1650 (b, d) cells transduced with retrovirus designed to overexpress either wild-type (a, b) or mutant (T34A) DARPP-32 (c, d) were transfected with GFP (control), kinase-dead (KD), full-length (FL), and constitutively active (CA) IKKα cDNAs were lysed using 1× RIPA buffer supplemented with protease inhibitors only. Equal amounts of proteins (500 ng) were immunoprecipitated using anti-PP1α antibodies. Immunoprecipitated cell lysates were subjected to in vitro phosphatase assays following incubation with either PP1α substrate or histone H1 peptide (control). Released phosphates in each reaction tube were determined by using a phosphate detection reagent. In vitro phosphatase experiments were repeated three times independently. Bar graphs represent the mean ± SEM of the three repeats, with each circle in a bar representing an independent experiment. A value of P ≤ 0.05 was considered significant, ns not significant, one-way ANOVA followed by Dunnett’s test. e–h Immunoprecipitated HCC827 (e, g) and H1650 (f, h) cell lysates separated with 4–20% SDS-PAGE were subjected to western blotting using anti-PP1α antibodies. Input cell lysates were blotted with antibodies against IKKα, DARPP-32, and α-tubulin (loading control).