Anti flag agarose affinity gel

ATM kinase was purified as described in Lashgari et al (2019 (link)) with slight modifications. Briefly, 8 L of K562 cells stably expressing near physiological levels of 3×FLAG-Twin-Strep-tagged ATM from the AAVS1 safe harbor were established as described previously (Dalvai et al, 2015 (link)). These cells were cultured in RPMI medium supplemented with 10% NBCS and 0.5 μg/mL puromycin. FLAG immunoprecipitations with anti-FLAG agarose affinity gel (Sigma M2) were performed on nuclear extracts followed by elution with 3×FLAG peptide, followed by Strep immunoprecipitation with Strep-Tactin XT 4Flow agarose beads (IBA), and eluted with D-biotin (100 mM D-biotin is prepared in the following buffer: 40 mM Hepes pH 7.9, 150 mM KCl, NaOH to dissolve and mixed with 1:1 ratio of the modified elution buffer: 20 mM Hepes pH 7.9, 150 mM KCl, 20% glycerol, 0.2% Tween-20, 2 mM DTT, and supplemented with 10 mM sodium butyrate, 10 mM β-glycerophosphate, 1 mM PMSF, 5 mM NaF, 100 µM orthovanadate, 2 µg/mL leupeptin, 2 µg/mL pepstatin, and 5 µg/mL aprotinin added fresh). The purified eluted fractions were loaded on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and visualized by silver staining.

Anti-GAPDH antibodies, anti-Flag agarose affinity gels, and anti-HA agarose affinity gels were supplied by Sigma–Aldrich (Shanghai, China). Mouse anti-Flag-horseradish peroxidase (HRP), anti-HA-HRP and anti-Myc-HRP antibodies were purchased from Roche (Basel, Switzerland). HRP-conjugated goat anti-mouse IgG (H + L) was obtained from Proteintech Group, Inc. (Rosemont, IL, USA).

The co‐immunoprecipitation was performed as previously described (Bowman et al., 2008) without crosslinking and with a few other notable differences. Cell cultures were grown to exponential phase in 1 l of PYE medium, then pelleted. Pellets were washed and re‐suspended in co‐immunoprecipitation buffer [20 mM HEPES pH 7.5, 100 mM NaCl, 20% glycerol, Pierce Protease Inhibitor Tablet (1 tablet/l)]. The cell suspension was passed through a French press at 16 000 psi three times to achieve lysis. Membranes were solubilized by the addition of IGEPAL CA‐630 (1%), sodium deoxycholate (0.5%) and 2 mM EDTA. 3xFLAG‐FzlA was then immunoprecipitated using anti‐FLAG affinity agarose gel (Sigma), and bound proteins were eluted using excess FLAG peptide (Sigma). Samples were subjected to immunoblot analysis (see antibodies and immunoblotting section below).

HEK293 cells were transfected with FLAG or Halo-tagged expression vectors. After 2–3 days, FLAG-tagged protein-expressing cells were lysed in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl at pH 7.5, 0.2% deoxycholate, and 0.5% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany); anti-FLAG affinity agarose gel (A2220, Sigma) was added and the mixture was incubated overnight at 4°C. The gel was washed thoroughly with PBS and FLAG-tagged protein was eluted by adding FLAG peptide. The Halo-tagged protein was purified using HaloLink resin (G1914, Promega), according to the manufacturer’s protocol.