Arpp19 ablation phenotypes are rescued by inhibiting the PP2A phosphatase and by the ectopic expression of WT ARPP19, but not of the ARPP19S62A mutant. (A) GFP and CRE mitotic Arpp19^Lox/Lox MEFs obtained by nocodazole shake-off were lysed and analyzed by Western blotting (left) with the indicated antibodies (p-S-CDK, antibody to detect phosphorylation on serine residues by CDK) or by immunofluorescence (right), and the total p-S-CDK immunofluorescence signal was quantified. (B) CDK1 activity was assessed by IP in the indicated mitotic MEF lysates followed by histone H1 phosphorylation. Total cell signal was quantified. (C) Top: ENSA protein levels were measured in GFP and CRE MEF lysates and ENSA IPs by Western blotting. Bottom left panel: ARPP19/ENSA phosphorylation at the GWL site was measured by immunofluorescence. Bottom right: Total immunofluorescence signal was measured in control and Arpp19^Δ/Δ MEFs using ImageJ and is represented as the means ± SEM. (D) As for C, except that Arpp19^Δ/Δ cells ectopically express HA-tagged WT human ARPP19 or the S62A mutant. (E) Quantification of the phenotypes observed in MEFs from D. Data pooled from two different experiments. (F) GFP or CRE MEFs were incubated with 5 nM OA and used for immunofluorescence. Phenotypes were counted and are represented. Bars, 10 µm.