Pima513244

Harvested cells were lysed for 40 min on ice in IP lysis buffer (0.75% CHAPS, 10% (vol/vol) glycerol, 150 mM NaCl, 50mM Tris pH 7.5) freshly supplemented with protease inhibitors. Supernatants were diluted to adjust the CHAPS concentration to 0.375%. Whole cell lysates (3 to 5 mg total protein) were incubated with anti-IgG rabbit (Millipore, N101), anti-IgG mouse (Millipore, N103), anti-DNA-PKcs (Thermo Fisher, PIMA513244 or Invitrogen, MA5-132238), anti-SIRT2 (Abcam, ab67299) or anti-GFP antibody (Abcam; Ab6556). Protein G beads, A agarose beads (Invitrogen), or FLAG conjugated beads (Sigma A2095) were used to immunoprecipitate antibody bound protein. Complexes were washed 4 times with IP washing buffer (0.375% CHAPS, 10% glycerol, 150 mM NaCl, 50 mM Tris pH 7.5) supplemented with protease inhibitors.

Harvested cells were lysed for 30 min on ice in Nonidet P-40 buffer (200 mM NaCl, 1% Nonidet P-40,50 mM Tris·HCl pH 8.0) or IP lysis buffer (0.75% CHAPS, 10% (vol/vol) glycerol, 150 mM NaCl, 50mM Tris pH 7.5) freshly supplemented with protease inhibitors. Protein samples were resolved by SDS-PAGE and probed with indicated antibodies: anti-SIRT2 (Abcam ab67299 or Santa Cruz sc-20966), GAPDH (Santa Cruz sc-25778 or sc-47724), FLAG (Sigma F4042), GFP (Abcam Ab6556), anti-DNA-PKcs (Thermo Fisher, PIMA513244 or Invitrogen, MA5-132238), Artemis (Abcam, ab3834), anti-Artemis phospho Ser516 (Genetex, GTX32292), XRCC4 (Thermo Fisher Scientific, MA5-24383), XRCC4 phospho Ser260 (Thermo Fisher Scientific, PA5-64731), Cyclin A (BD BioScience, 611268) and α-Tubulin (Sigma, T6074). Detection was performed with the Odyssey system.