Fig. 2: Ammonium sulfate precipitation separates S109-phosphatase from PKA, PP1 and PP5.

a Western blot analysis of various S/T phosphatases sensitive to OA in lysates from prophase (Pro) or metaphase II (MII) oocytes using specific antibodies directed against catalytic subunits of PP1, PP2A (PP2A-C), PP4, PP5, PP6, and PP2A-regulatory subunit A (PP2A-A), B55δδ, and B56ε. The experiment was repeated 3 times with similar results. b–e Prophase extracts supplemented or not with PKI were precipitated by serial addition of ammonium sulfate (AS) as indicated. (–): Starting extracts without AS. Pellets were recovered and used for enzymatic assays and western blots with phospho-S109-Arpp19 and GST antibodies. S109-phosphatase activity was assayed using pS109-GST-Arpp19 (pS: phosphorylated substrate): one representative experiment (b) and quantifications of S109 phosphorylation from 3 independent experiments (c). d–e PKA activity was assayed using GST-Arpp19 (npS: non-phosphorylated substrate): one representative experiment (d) and quantifications of S109 phosphorylation from 3 independent experiments (e). For quantifications, data are presented as mean (red bars) ± SEM. Each dot represents one experiment. arb. units: arbitrary units. f Western blot analysis of initial extracts (–) and AS precipitates using specific antibodies directed against catalytic subunits of PP1, PP2A (PP2A-C), PP4, PP5, PP6, PKA, and against PP2A scaffold subunit A (PP2A-A) and PKI. The experiment was repeated 3 times with similar results. kDa: kiloDalton. Source data are provided as a Source Data file.