M2 agarose resin

Trr complex was reconstituted by co-transfecting Sf9 cells with the cocktail of five baculoviruses expressing Trr-WinSET, hRBBP5, hASH2L, hWDR5, and hDPY30, respectively, and purified with M2 agarose resin (sigma-aldrich). All five components are N-terminally FLAG-tagged. HMTase assay was performed in 20μL of 50mM Tris-HCl pH8.8, 20mM KCl, 5mM MgCl2, 0.5mM DTT at 37°C for 1hr with 1μg of recombinant histone H3 (NEB), 200μM of S-adenosyl-methionine, and near equal amounts of Trr + hWRAD.

For kinase assays Hela (female) cells with stable expression of wildtype or C312S flag-tagged CDK7 were first treated with DMSO, YKL-5-124, or YKL-5-167 for 6 hours. Cells were then harvested by lysis in 50 mM TrisHCl pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, Protease Inhibitor (Roche), and Phosstop Phosphatase Inhibitor (Roche). CDK7-FLAG was immunprecipitated from lysates using M2-agarose resin (Sigma, A2220). Precipitated proteins were washed with lysis buffer 4 times, followed by 2 washes with kinase buffer (40 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5% glycerol) and subjected to in vitro kinase assays at 30°C for 45 minutes using 1 μg of the large subunit of RNAPII (RPB1) as substrate and 25 μM ATP and 10 μCi of 32P ATP. Reactions were run on SDS-PAGE gel, fixed and stained with coomassie reagent, and exposed to phospho-imager screen before being scanned on a TYPHOON imager (GE Healthcare Life Sciences).