Prpas33

Cells on coverslips were washed with PBS and fixed in 4% PFA/2% sucrose solution for 15 min. The coverslips were washed again with PBS and then Triton extracted (0.5% Triton X-100 in PBS) for 4 min. Cells were incubated with their respective antibodies for 30 min at 37°C followed by incubation with secondary antibodies (FITC or Rhodamine) for 30 min at 37°C. Primary antibodies used in IF studies were RPA34 (Cal Biochem; NA18; 1:100), 53BP1 (Bethyl; A300-272A; 1:2,000), g-H2AX (Millipore; 05–636; 1:5,000), RPA32 (Thermo Fisher Scientific; PA5-22256; 1:400), pRPAS33 (Sigma; PLA0210-100 μl; 1:1,500), and BRCA1 (Upstate; 07–434; 1:400). Coverslips were mounted using mounting medium (DAPI). Images were acquired with an Axio Imager.M2 (Carl Zeiss) equipped with an Axiocam 506 color camera, controlled by Zen software.

Whole-cell extracts were prepared by lysing cells in NETN450 lysis buffer (450 mM NaCl, 20 mM Tris-HCl, pH 7.8, 0.5% NP-40, 1 mM EDTA, and dH₂O). For nuclear extracts, cells were lysed in Protein Extraction (PEB; 0.5% Triton X, 20 mM Hepes, pH 7, 100 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, and dH₂O) on ice for 20 min followed by spinning at 5,000 rpm for 10 min to remove the cytoplasmic extract. Cell pellets were washed once with PBS followed by lysing in NETN 400 lysis buffer (400 mM NaCl, 20 mM Tris-HCl, pH 7.8, 0.5% NP-40, 1 mM EDTA, and dH₂O) for 1 h at 4°C to generate the nuclear extract. All lysis buffers were supplemented with protease inhibitor and phosphatase inhibitor. Antibodies used for Western blot were RFWD3 (Bethyl; A301-397A; 1:2,500), BRCA2 (Bethyl; A300-005A; 1:3,000), SD118 (Calbiochem; OP107; 1:2,500), GAPDH (Santa Cruz; SC-25778; 1:4,000), GAPDH (BioLegend; 919501; 1:4,000), RAD51 (Santa Cruz; SC-8349; 1:2,500), HA (BioLegend; 901514; 1:3,000), LaminB1 (Cell Signaling; 12596; 1:3,000), pRPA32 S4/S8 (Bethyl; A300-245A; 1:2,500), pRPA S33 (Sigma; PLA0210; 1:2,500), RPA34 (Calbiochem; NA18; 1:3,000), α-Tubulin (Santa Cruz; SC-5286; 1:3,000), Mre11 (Genetex; GTX70212; 1:3,000), and SMARCAL1 (Bethyl; A301-616A; 1:2,500).