Kinase buffer

GST-tagged MST2 or MST2-S385A (cloned in pGEX-5X-1) was bacterially expressed and purified on GSTrap FF affinity columns (GE Healthcare) following the manufacturer’s instructions. GST-MST2 (1 μg) was incubated with 5-10 U recombinant CDK1/cyclin B complex (New England Biolabs) or 50-100 ng CDK1/cyclin B (SignalChem) in kinase buffer (New England Biolabs) in the presence of 5 μCi γ-³²P-ATP (3000 Ci/mmol, PerkinElmer) as we previously described [15 (link)]. Active CDK2, CDK5, p38, JNK1, JNK2, MEK1, ERK1, and PLK1 kinases were also purchased from SignalChem.

GST-tagged MARK2, MARK2-3A (amino acids 388-788), HDAC4, HDAC4-3A (amino acids 200-680) and MST2-KD (kinase dead) proteins were bacterially expressed and purified on GSTrap FF affinity columns (GE Healthcare) following the manufacturer’s instructions. The GST-MARK2 proteins (0.5-1 μg) were incubated with recombinant 100 ng of CDK1–Cyclin B1 (SignalChem) in kinase buffer (New England BioLabs) in the presence of 10 μCi of [γ-³²P]ATP (3000 Ci/mmol, Perkin Elmer Life Sciences). GST-MST2-KD and GST-HDAC4 proteins (1 μg) were also used as substrates for MARK2 (150 ng) phosphorylation. Active MARK2, CDK5–p25, MEK1, ERK1, JNK1, JNK2, and p38α kinases were purchased from SignalChem. Phosphorylation (³²P incorporation) was visualized by autoradiography followed by Western blotting or detected by phospho-specific antibodies.

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.