Immunoprecipitation experiments were performed using 3×106 293T cells. IP buffer (0.5% Triton X-100, 50mM Tris HCl pH 8.0, 150 mM NaCl, EGTA 1 mM) supplemented with phosphatase, protease inhibitors and benzonase was used for cells lysis. Two mL of lysate were incubated overnight at 4°C with 20 μl of Anti-Flag M2 magnetic beads (Sigma) or Anti-RPA32 conjugated Dynabeads (2μg of MABE285 anti-RPA34–20 mouse (Millipore) with 40μl of Dynabeads protein G (Invitrogen). After extensive washing in IP buffer, proteins were released in 2X Laemmli buffer buffer and subjected to Western blotting.
Blots were incubated with primary antibodies: rabbit anti-FLAG (Sigma-Aldrich); rabbit anti-RPA70 (Genetex); mouse anti-RPA34–20 (Millipore); rabbit anti-pS440/467WRN (Abgent; custom-made); rabbit anti-Lamin B1 (Abcam); rabbit anti-RAD51 (Santa Cruz); rabbit anti-GST (Calbiochem). Blots were detected using the Western blotting detection kit WesternBright ECL (Advansta) according to the manufacturer’s instructions. Quantification was performed on scanned images of blots using Image Lab software, and values shown on the graphs represent normalization of the protein content evaluated through Lamin B1 or Immunoprecipitated protein immunoblotting.
Blots were incubated with primary antibodies: rabbit anti-FLAG (Sigma-Aldrich); rabbit anti-RPA70 (Genetex); mouse anti-RPA34–20 (Millipore); rabbit anti-pS440/467WRN (Abgent; custom-made); rabbit anti-Lamin B1 (Abcam); rabbit anti-RAD51 (Santa Cruz); rabbit anti-GST (Calbiochem). Blots were detected using the Western blotting detection kit WesternBright ECL (Advansta) according to the manufacturer’s instructions. Quantification was performed on scanned images of blots using Image Lab software, and values shown on the graphs represent normalization of the protein content evaluated through Lamin B1 or Immunoprecipitated protein immunoblotting.