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. 2022 Jun 13;41(14):e110611. doi: 10.15252/embj.2022110611

Figure EV1. Perphenazine, iHAP1 and DT‐061 do not activate PP2A complexes in vitro .

Figure EV1

  • A
    Titration experiment of different PP2A holoenzyme preparations to determine appropriate concentration to use in enzymatic assays. Experiment was performed twice using the concentrations of enzyme stated in the figure. Shown is one representative experiment.
  • B–E
    In vitro dephosphorylation assays using model phosphopeptides and purified PP2A holoenzymes. The phosphopeptides were incubated with specific PP2A holoenzymes together with PPZ, iHAP1 or DT‐061 at the indicated concentrations. The reaction was stopped after 15 min, and the amount of phosphate released was measured. In (E) substrates are BRCA2 WT: P(pS)QKAEITELSTILEESGSQW and BRCA2 2A: P(pS)QKAEITEASTALEESGSQW. Mean and SD from three technical replicates of one out of three independent experiments is shown. Experiment in (E) is representative of two independent experiments.
  • F
    Injection of PPP2R1A (300 μM) into the ITC buffer (5% DMSO) used for the DT‐061, iHAP experiments. The experiments were performed at 25°C. No significant effects due to dilution of protein (control experiment for Fig 1B)