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PP2A immunoprecipitation phosphatase assay

YH Yuka Hotokezaka
IK Ikuo Katayama
TN Takashi Nakamura
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Assays for PP2A phosphatase activity bound to the ATM protein were performed by using a commercially available assay kit (Millipore). Briefly, FLAG-tagged ATM precipitates were washed with Ser/Thr assay buffer and incubated at 30 °C for 10 min after adding 750 μM phosphopeptide (K-R-pT-I-R-R). The precipitates were then centrifuged briefly, and Malachite Green Phosphate Detection Solution (100 μL, Millipore) was added to the supernatant (25 μL), and let color develop for 10–15 min at room temperature. The protein phosphatase activity was determined by reading the solution at 650 nm with the microplate reader (Wallac 1420 ARVOsx).

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