Myc trap magnetic beads

Cells were collected 24 hours after transfection and lysed in lysis buffer [0.5% (v/v) NP-40, 10 mM tris-HCl (pH 7.5), 0.5 mM EDTA, and 150 mM NaCl] supplemented with a protease and phosphatase inhibitor cocktail (Cell Signaling Technology). After centrifugation for 10 min at 14,000g, supernatants were collected and incubated with anti-FLAG magnetic beads (Sigma-Aldrich) or Myc Trap magnetic beads (Chromotek) overnight at 4°C. Beads were washed three times with cold lysis buffer and eluted with 3×FLAG peptide (Sigma-Aldrich) or Laemmli sample buffer, respectively. For endogenous TBK1 immunoprecipitation assays, cell lysates were obtained in lysis buffer [20 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, leupeptin (1 μg/ml), and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Cell extracts were incubated overnight at 4°C, with 1 μg of anti-TBK1 antibody and protein A magnetic beads (Cell Signaling Technology). The next day, beads were washed with cold lysis buffer and resuspended in Laemmli sample buffer. All samples were heated at 95°C for 10 min.

The transfected or infected cells were washed with DPBS three times and lysed with Lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NonidentP40) containing protease inhibitors (cOmplete; Roche), 1 mM phenylmethylsulfonyl fluoride (Nacarai), 10 mM N-ethylmaleimide (NEM, Sigma) as a DUB inhibitor and 10 μM MG132 (Calbiochem) as a proteasome inhibitor. After removal of cell debris with centrifugation, cell lysates were incubated with FLAG M2 magnetic beads (Sigma), RFP-Trap magnetic beads (chromotek) or Myc-Trap magnetic beads (chromotek) for 2 hr to overnight at 4°C. The beads were washed five times with wash buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100), and the bead-bound proteins were eluted by boiling in SDS sample buffer.

Cells were plated in 10 cm dishes and transfected the next morning with 6 μg GFP-RB plasmid, 6 μg myc-RAD51 plasmid, or the combination. Twenty-four hours later, cells were lysed in 1.0 mL RIPA buffer with protease and phosphatase inhibitor, rocked for 1 hour at 4°C and then spun down, and the pellet was discarded. A BCA assay was used to standardize protein concentrations: approximately 100 mg was removed for input and approximately 1000 mg was used for the IP. Lysates were precleared by incubation with 20 μL of A/G Plus agarose beads (sc-2003) for 1 hour at 4°C; then, GFP-Trap Magnetic Beads (Chromotek, GTD-20), Myc-Trap Magnetic Beads (Chromotek, YTMA-20), or Binding Control Magnetic Agarose Beads (Chromotek, BMAB-20) were added to the cleared lysate, which was left to slowly rock overnight at 4°C. Beads were washed the next morning with RIPA buffer, and bound proteins were eluted in 50 μL RIPA buffer with 1× NuPAGE and 2% β-mercaptoethanol.