Sepharose cl 4b beads

Commercial antibodies used were as follows: anti-FLAG (Sigma), anti-CDYL (Sigma, HPA035578; for Western blotting), anti-CDYL (Abcam, ab5188), anti-BrdU (Abcam, ab6326), anti-RPA1 (ABclonal, A0990), anti-RAD51 (ABclonal, A6268), anti-WRN (ABclonal, A6855), anti-DNA2L (Abcam, ab2179), anti-BLM (Abcam, ab96488), anti-MRE11 (Cell Signaling Technology, 4847), anti-NBS1 (Cell Signaling Technology, 14956), anti-RAD50 (Cell Signaling Technology, 3427), anti-Myc, anti-hemagglutinin (HA), anti–β-actin (MBL), anti-PanKac (PTM, 101), anti-PanKcr (PTM, 502; for Western blotting), and anti-PanKcr–conjugated agarose beads (PTM, 503; for immunoprecipitation). A CDYL antibody that we generated previously (peptide antigen: KQKESTLTRTNRTSPNN; B&M) was used for immunoprecipitation. The polyclonal antibodies against RPA1 K88cr, RPA1 K379cr, and RPA1 K595cr were generated by immunizing rabbits with two synthetic crotonyl peptides corresponding to residues surrounding K88, K379, and K595 of RPA1, respectively. HU, CPT, and VP16 were from Sigma. Streptavidin agarose resins were from Thermo Fisher Scientific. Protein A/G and Sepharose CL-4B beads were from GE Healthcare Biosciences, and protease inhibitor mixture cocktail was from Roche Applied Science.

HCT116 BAX/BAK DKO cells were transfected with an empty cDNA3-mycBioID plasmid or cDNA3-mycBioID-BAX plasmids (wild-type, dC, 63–65A, dBH3) or YFP-tBid, YFP-Bim with and without BCL-x_L plasmid as indicated. When stated, cells were treated with 25 µM CLZ (Sigma-Aldrich) or solvent control (DMSO) for 4 h. Cells were harvested and washed with PBS prior to lysis in immunoprecipitation (IP)–solubilization buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 10% glycerol, and 0.1 mM EDTA) supplemented with 2% digitonin (MATRIX BioScience GmbH) and proteinase inhibitors. After 20 min incubation on ice, cells were passaged though a 1-mL syringe (Braun) with a 25G needle (Braun). The cell debris was cleared (15,000 × g, 10 min, 4 °C) and 2.5% of the supernatant were used as input. The lysis was incubated with washed protein A Sepharose cl-4B beads (GE Healthcare) and GFP antibody (Novus) or Myc beads (C-myc [9E10] Santa Cruz), at 4 °C overnight. The following day, the beads were washed five times with IP-solubilization buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 10% glycerol, and 0.1 mM EDTA), supplemented with 0,02% digitonin and proteinase inhibitors after centrifugation at 350 × g, 3 min, and 4 °C. Beads were boiled in SDS sample buffer, and input and IP samples were analyzed by Western blot.