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. 2022 Aug 24;119(35):e2210367119. doi: 10.1073/pnas.2210367119

Fig. 1.

Fig. 1.

Influence of phosphatase inhibitors on the binding of Cdc20 to APC/C in anaphase extracts from Xenopus eggs. (A) Effects of okadaic acid and calyculin A on the phosphorylation state of Cdc27 and Cdc20 in anaphase extracts. Interphase or anaphase extracts from Xenopus eggs were prepared as described under Materials and Methods. Anaphase extracts were incubated at 23 °C for 30 min with okadaic acid (Calbiochem #MBS459620) or calyculin A (Cell Signaling #CST-9902S) (1 mM, each), as indicated. DMSO solvent was at a final concentration of 0.5% (v/v) in all incubations. Where indicated, the sample was treated with lambda phosphatase (see Materials and Methods). Samples of 0.1 mL of extracts were subjected to SDS-PAGE and were immunoblotted as indicated. Numbers on the right indicate the position of molecular mass marker proteins. Ana., anaphase; Ok. ac, okadaic acid; Cal. A., calyculin A; lPP, lambda phosphatase. (B) Effects of phosphatase inhibitors on the binding to APC/C of total and T79-phosphorylated Cdc20. Anaphase extracts from Xenopus eggs were incubated with or without phosphatase inhibitors as in A, and then were subjected to immunoprecipitation with anti-Cdc27 antibodies or with nonimmune IgG, as indicated. Immunoprecipitation was carried out as described under Materials and Methods, except that beads were washed four times with buffer A that contained 0.3 M NaCl and 0.1% (vol/vol) NP-40 and then twice with buffer A. Where indicated, samples were treated with lambda phosphatase (“lPP”), as described under Materials and Methods. Samples of immunoprecipitates derived from 5 mL extracts (lanes 4–12) or 0.1 mL of unfractionated extracts (lanes 1–3) were subjected to immunoblotting with antibody directed against Xenopus Cdc20 (Upper) or against its T79-phosphorylated derivative (Center). (Lower) Immunoblot form Cdc27 bait in same immunoprecipitates. Asterisks, cross-reacting material; arrows, position of pT79-Cdc20. (C) Quantitation of data similar to B. Data from three experiments similar to B were quantified. APC/C-bound total Cdc20 or pT79-Cdc20 were expressed as the percentage of each in unfractionated extracts.