Monoclonal anti ha agarose beads

Flag-ATR–HA-ATRIP complexes were purified from HEK293T cell nuclear extracts with monoclonal anti-HA agarose beads (Sigma-Aldrich, A2095). Beads were washed three times with TGN buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 1% Tween20, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mM sodium orthovanadate, 10 mM β-glycerol phosphate, 1 mM NaF, 0.5 mM DTT) and once with TGN buffer containing 500 mM LiCl. Beads were then washed twice with kinase buffer (10 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 10 mM MnCl₂, 50 mM β-glycerol phosphate, 1 mM DTT). ATR-ATRIP bound beads were incubated with GST, a GST-AAD, MBP-AAD, FKBP F36V-AAD, or untagged AAD, a GST- or untagged substrate, and [γ-³²P]ATP for 20 min at 30 °C. Reactions were stopped by addition of 2× SDS sample buffer prior to protein separation by SDS-PAGE and detection by Coomassie staining. Substrate phosphorylation was detected by phosphoimaging. In experiments performed with FKBP F36V-AADs, the FKBP F36V-AADs were incubated with DMSO or 5 μM AP20187 (Clontech, 635058) for 1 h at room temperature prior to beginning kinase reactions. Kinase reactions also contained either DMSO or 5 μM AP20187.

293 T whole cell extracts were prepared using lysis buffer containing 1%TX-100, 50 mM HEPES (pH 7.4), 138 mM KCl, 4 mM NaCl, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 1 mM EGTA pH 8, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, and protease inhibitors (Roche cat # 11697498001 or Thermo Scientific cat # 88265) as previously described (Lamia et al.^{40 (link)}). ASF cell extracts were prepared from RIPA buffer containing 1% TX-100, 147 mM NaCl, 12 mM sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and protease inhibitors (Thermo Scientific cat # 88265). Antibodies used for immunoprecipitation were anti-FLAG M2 agarose beads (Sigma cat # A2220) and monoclonal anti-HA agarose beads (Sigma cat # A2095). Antibodies for Western blot were anti-FLAG polyclonal (Sigma cat # F7425), anti-HA polyclonal (Sigma cat # H6908), anti-MYC polyclonal (Sigma cat # C3956), anti-SKP1 (BD Biosciences cat # BDB610530), anti-CUL1 (Life Technologies cat # 71–8700), anti-αTubulin (Sigma cat # T5168), anti-βactin (Sigma cat # A1978), anti-AFG3L2[N1N2] (GeneTex cat # GTX102036), and CRY1-CT and CRY2-CT as described (Lamia et al., 2011).