Kinase assay

For the kinase assay, 293T cells transfected with empty vector (EV) or HA‐ATR were lysed in TGN buffer (150 mM NaCl, 50 mM Tris pH 7.4, 10% glycerol, 1% Tween‐20) containing protease inhibitors and phosphatase inhibitors (1 mM NaF, 50 mM β‐glycerophosphate, 1 mM sodium vanadate) and 1 mM DTT. The lysates were sonicated followed by centrifugation at 20,000 g for 10 min. Immunoprecipitation of HA‐ATR was performed by incubating the lysates with HA‐agarose (Sigma) for 2 h at 4°C on a rotator. The beads were washed three times with TGN buffer and two times with TGN buffer supplemented with 500 mM LiCl and finally two times with kinase buffer (10 mM Hepes pH 7.5, 50 mM NaCl, 50 mM β‐glycerophosphate, 10 mM MgCl₂, 1 mM DTT). The HA‐ATR binding beads were incubated with GST‐PALB2‐N in a reaction mix containing kinase buffer, 1 mM DTT, 50 mM β‐glycerophosphate, 150 μM ATP and 10 mM MnCl₂. 10 μCi [γ‐³²P]‐ATP was added to the mix and the reaction was incubated at 30°C for 40 min with gentle shaking. Laemmli sample buffer was added, and the samples were resolved with SDS–PAGE. The gel was either stained with InstantBlue Coomassie stain (Expedeon) and dried, or the resolved proteins were transferred to a nitrocellulose membrane. The radioactive signal was detected using Fujifilm Phosphorimager.