Atr antibody

The cytoplasm of A549 cells was isolated after UV treatment. ATR antibody (Bethyl Labs) was added for pull-down overnight. IPed ATR protein was subjected to a 0.6 M NaCl wash to remove associated proteins and suspended in kinase buffer (New England Biolabs). Cdk1-Cyclin B (New England Biolabs, P6020S) was added and incubated for 1 hr at 30°C to phosphorylate the isolated IPed ATR. After centrifugation and washing of the bead-bound ATR purified Pin1 was added to the phosphorylated ATR and incubated for 1 hr at 30 °C. SDS-loading buffer was added and ATR isomerization assayed by 3–8% TA SDS-PAGE and WB. Alternatively, to measure Pin1 binding to phosphorylated ATR the IPed ATR was incubated first with Cdk1 for 1 hr at 30°C, washed thrice, and purified Pin1 added for a 2 hr incubation at 4°C. The ATR-beads were washed three times and suspended in SDS loading buffer for WB analysis. To assess tBid binding to ATR-H following UV treatment of A549 cells, the cytoplasmic fraction or isolated mitochondria was collected. ATR antibody (Bethyl Labs) was added for pull-down overnight. IPed ATR-beads were washed three washes in Co-IP wash buffer (50 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Tween-20). The beads were suspended in 1x SDS loading buffer, boiled at 95°C for 5 min and analyzed by WB.