Antibodies and immunoblotting

To extract proteins from S. cerevisiae, harvested cells were washed twice with PBS and resuspended in 100 μl of lysis buffer with protease inhibitors and phosphatase inhibitors [150 mM NaCl, 50 mM tris (pH 7.5), 0.15% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, leupeptin (1 μg ml⁻¹), pepstatin (1 μg ml⁻¹), 10 mM sodium fluoride, 20 mM β-glycerolphosphate, 10 mM sodium orthovanadate, and 10 mM sodium pyrophosphate]. Cells were disrupted by bead beating using 0.5-mm glass beads. Lysates were clarified by centrifugation at 14,000 revolutions per minute (RPM) for 10 min on a 4°C precooled microcentrifuge. Protein concentration was determined by Bradford assay. Protein samples were mixed with 6× SDS sample buffer and boiled for 5 min at 95°C. SDS-PAGE was performed with 8% separating gel. For Phos-tag SDS-PAGE, 50 μM Phos-tag and 100 μM MnCl₂ were mixed with 6% separating gel. For λ phosphatase assay, lysate was incubated with 600 U of λ phosphatase (New England Biolabs), 1× NEBuffer for protein metallophosphatases, and 1 mM MnCl₂ at 30°C for 2 hours.

Immunoblotting was performed using the following antibodies: an anti–phospho–mitogen-activated protein kinase (MAPK)/CDK substrates (Cell Signaling Technology, 23255) for phospho-Rad51, an anti-Rfa1 antibody (Abcam, ab221198) for intact Rfa1, an anti-Rad51 antibody (Abcam, ab63798) for intact Rad51, a horseradish peroxidase (HRP)–conjugated anti–influenza hemagglutinin (HA) antibody (Santa Cruz Biotechnology, SC-7392 HRP) for Rad52-HA, an anti-myc antibody (Santa Cruz Biotechnology, SC-40) for Rad52-myc, an HRP-conjugated anti-GFP antibody (Santa Cruz Biotechnology, SC-9996 HRP) for GFP-tagged proteins, an anti-mouse immunoglobulin G (IgG) antibody (Sigma-Aldrich, A9044) for Cdc28-TAP (tandem affinity purification), an anti–glutathione S-transferase (GST) antibody (Santa Cruz Biotechnology, SC-138) for GST-tagged proteins, an polyclonal anti-GFP antibody (Rockland, 600-103-215) for VN (the N-terminal fragment of Venus)/VC (the C-terminal fragment of Venus)–tagged Rad52, and an anti-hexokinase antibody (Rockland, 100-4159) for endogenous hexokinases.