In vitro kinase assays

In vitro Chk1 or TLK1 kinase assay was performed in kinase buffer D (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 25 µM ATP, 8 µCi of [γ³²P] ATP). 200 ng of recombinant Chk1 (#7735; Biovision) or immunoprecipitated HA-TLK1 was incubated with 1 µg recombinant His-ASF1A for 30 min at 30ºC and the reaction stopped by addition of Laemmli buffer. UCN-01 was directly added to the reaction tube at 100 nM concentration to inhibit Chk1. Reactions were separated on 13% SDS-polyacrylamide gels. To obtain HA-TLK1 protein, HA-TLK1 expressing plasmids were transfected into HEK293T cells, and the cells were lysed in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM MgCl2, 10 mM NaF, 0.1 mM sodium vanadate, and protease inhibitors) after 48 hr. 150 µg of lysate was mixed and incubated with EZview Red Anti-HA Affinity beads (E6779; Sigma) for 2 hr, and the beads were washed twice with lysis buffer followed by twice with kinase buffer. In vitro ATM kinase assay was performed using 200 ng of recombinant ATM (14–933; Sigma) in kinase buffer E (10 mM Tris-HCl (pH 7.5) 50 mM KCl 10 mM MgCl₂ 10 mM MnCl₂ 1 mM DTT 200 µM ATP, 4 µCi of [γ³²P] ATP).