Fig 1: Biochemical isolation of S109-phosphatase from prophase oocyte extracts - Separation of fractions 7 and 8 from the Mono Q column with Phenyl-Superose and Superose 12 columns.Continuation of experiment illustrated in Fig. 3. “pS”: phosphorylated pS109-GST-Arpp19 substrate. “In”: input sample loaded on the column. kDa: kiloDalton. arb. units: arbitrary units. a Phenyl-Superose. Fractions 7 and 8 from the Mono Q column (see Fig. 3c) were pooled and loaded on the column. Elution profile of S109-phosphatase activity after Phenyl-Superose column and western blot analysis of fractions 1–14 with antibodies directed against the catalytic subunits of PP1, PP2A (PP2A-C), PP4, PP5, and PP6, PP2A scaffold subunit (PP2A-A) and PP2A regulatory subunits B55d and B56e. b Superose 12. Fraction 10 and 11 from the Phenyl-Superose column (see a) were pooled and loaded on the column. Elution profile of S109-phosphatase activity after Superose 12 column and western blot analysis of fractions 3–14 with antibodies directed against the catalytic subunits of PP2A (PP2A-C) and PP5, PP2A scaffold subunit (PP2A-A) and PP2A regulatory subunits B55d and B56e. *non-specific protein recognized by the anti-PP5 antibody. Source data are provided as a Source Data file.
Fig 2: Biochemical isolation of S109-phosphatase from prophase extracts - Analysis of the output fractions of Uno Q and Mono Q columns.a Protocol of S109-phosphatase biochemical isolation. 20,000 prophase oocytes were lysed, centrifuged and fractionated by 4 successive steps of chromatography: two anion exchange columns (Uno Q and Mono Q), one hydrophobic column (Phenyl-Superose) and one size exclusion column (Superose 12). PKI-supplemented extracts from prophase oocytes were fractionated by Uno Q (b) and then Mono Q (c). S109-phosphatase activity was assayed in each fraction using pS109-GST-Arpp19 as a substrate (pS: phosphorylated substrate). S109 phosphorylation of GST-Arpp19 (pS109) and total GST-Arpp19 (gstArpp19) were analyzed by western blot using respectively phospho-S109-Arpp19 and GST antibodies. Fractions were western blotted with antibodies against the catalytic subunits of PP1, PP2A (PP2A-C), PP4, PP5 and PP6, PP2A scaffold subunit A (PP2A-A) and PP2A regulatory subunits B55d and B56e. *: non-specific protein recognized by the anti-B56e antibody. “C”: control extracts before PKI addition. “In”: input sample supplemented with PKI and loaded on the column. “FT”: flow-through. arb. units: arbitrary units. b Uno Q. FT and elution profile (fractions 1–3) of S109-phosphatase activity after Uno Q column and western blot analysis of fractions 1 to 6 of FT. (c) Mono Q. Fractions 2 to 5 of the Uno Q column FT (see b) were pooled and loaded on the column. Elution profile of S109-phosphatase activity after Mono Q column and western blot analysis of fractions 1 to 13. kDa: kiloDalton. Source data are provided as a Source Data file.
Fig 3: Biochemical isolation of S109-phosphatase from prophase extracts—Separation of fraction 5 from the Mono Q column with Phenyl-Superose and Superose 12 columns.Continuation of experiment illustrated in Fig. 3. “pS”: phosphorylated pS109-GST-Arpp19 substrate. “In”: input sample loaded on the column. kDa: kiloDalton. arb. units: arbitrary units. a Phenyl-Superose. Fraction 5 from the Mono Q column (see Fig. 3c) was loaded on the column. Elution profile of S109-phosphatase activity after Phenyl-Superose column and western blot analysis of fractions 3–15 with antibodies directed against catalytic subunits of PP2A (PP2A-C) and PP4, PP2A scaffold subunit A (PP2A-A) and PP2A regulatory subunit B55d. b Superose 12. Fraction 11 from the Phenyl-Superose column (see a) was loaded on the column. Elution profile of S109-phosphatase activity after Superose 12 column and western blot analysis of fractions 2–13 with antibodies directed against PP2A scaffold subunit (PP2A-A), PP2A catalytic subunit (PP2A-C) and PP2A regulatory subunit B55d. Source data are provided as a Source Data file.
Fig 4: 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), B55dd, and B56e. 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.
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