Slik isoforms are overphosphorylated in absence of PP2A_STRIPAK without affecting its kinase activity. (A) Schematic representation of Slik mRNA splicing variants (adapted from http://flybase.org/reports/FBgn0035001) and their expected molecular weight (kD). The longer isoforms (Slik-L) code for an additional C-terminal NCD compared with the shorter isoforms (Slik-S). dsRNA designed against all Slik isoforms or specific to Slik-L are indicated. (B and F) S2 cells treated as indicated were analyzed by Western blotting with antibodies against Slik, p-moesin, moesin, or β-actin. The upper bands correspond to Slik-L and lower bands to Slik-S isoforms. (C and G) Relative Slik or relative p-moesin levels (normalized as indicated) were quantified in three or four independent experiments (circles). Data are represented as mean ± SD normalized on nontarget (NT) dsRNA, one-way ANOVA with the following P values: NS, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D) S2 cells treated as indicated were lysed either in the absence (−) or presence (+) of λ-PPtase. Protein extracts were then analyzed by Western blotting using antibodies against Slik or β-actin. (E) Representation of the line scans of Western blots on Slik signals (bottom to top). The shifted Slik-L or Slik-S signals are indicated with arrows. (H) Western blot of S2 cells, treated as indicated, with antibodies against p-moesin, moesin, Slik, or β-actin (upper rows). Immunoprecipitated Slik from the treated cell lysates was incubated with purified moesin C-ERMAD-GST in a kinase reaction with [γ-³²P]ATP. The resulting samples were subjected to SDS–PAGE, and the dried Coomassie-stained gel was autoradiographed (middle and bottom rows). (I) Autoradiographed signals of moesin C-ERMAD(P₃₂)-GST were quantified in three independent experiments (circles). Data are represented as mean ± SD normalized on nontreated dsRNA, one-way ANOVA with the following P values: NS, P > 0.05; **, P < 0.01; ****, P < 0.0001. NT, non-target; F-ABD, F-Actin Binding Domain.