Precast gels

Protein extraction, immunoprecipitation and western blotting were performed as described (Nozawa et al., 2010 (link)). For whole cell protein extraction, cells were lysed in SDS buffer (62.5 mM Tris-HCl pH6.8, 1% SDS, 10% glycerol, 0.625% 2-Mercaptoethanol). The extracts from 1.0 × 10⁵ cells were separated by precast gels (FUJIFILM Wako) for Figures 1B, S1B, S3, S4A, and S5A or house-made gels for Figure 3B and S2B. Transfer of proteins onto PVDF membranes and incubation with primary/secondary antibodies were performed by standard procedures. The blots probed with secondary antibodies conjugated with horse radish peroxidase were exposed with ImmunoStar (FUJIFILM Wako) and imaged with LumiVision PRO 400EX system (AISIN) or LuminoGraph I system (ATTO). Quantifications of bands intensity were done by ImageJ (Figure S4A).
For immunoprecipitation, cells were lysis with CSK buffer (0.5 M NaCl, 0.1% Triton X-100, 10 mM PIPES-NaOH pH 7.0, 300 mM sucrose, 1 mM MgCl₂, 1 mM EDTA) supplemented with 100 μM PMSF (phenylmethanesulfonylfluoride) and 2 μg/ml leupeptin, and centrifugated supernatant was used as nuclear extract. Anti-FLAG M2 Affinity Gel (Sigma-Aldrich) was used for immunoprecipitation of FLAG-tagged RIF1 complexes from the nuclear extracts.

Cells were lysed in RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) for 30 minutes on ice. The lysates were denatured in equal volumes of 2× sodium dodecyl sulfate (SDS) sample buffer (Bio-Rad, CA) containing 5% (volume per volume) 2-mercaptoethanol (Sigma-Aldrich, MO) and heated for 5 minutes at 95°C. Next, they were fractionated by SDS–polyacrylamide gel electrophoresis on precast gels (Wako, Richmond, VA) and transferred onto nitrocellulose membranes (Wako). Before incubation with primary antibodies, the blots were blocked in 5% dry nonfat milk in Tris (hydroxymethyl) aminomethane–buffered saline with 0.1% Tween for 60 minutes. For protein detection, primary antibodies detecting THBS-1, IKZF1, Meis2, damage-specific DNA binding protein 1 (DDB1), glutathione S-transferases, His (Abcam), cereblon (Merck), and β-actin (Cell Signaling Technology, MA) were used. Secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse heavy chain– (BioLegend) and light chain–specific antibodies (Jackson ImmunoResearch, PA). The blots were visualized using ECL Prime Western Blot Detection Reagent (GE Healthcare, IL).