Nbp1 31883

Protein was extracted from cells using a lysis buffer (40 mM NaCl, 25 mM Tris pH 8, 2 mM MgCl₂, 0.05% SDS, 2x Complete EDTA-free protease inhibitor (Roche), 0.4 µL mL⁻¹ benzonase) with protein concentration determined by Bradford assay, comparing to BSA standards to ensure loading of an equal mass of protein into each lane. Protein and Laemmli buffer were heated to 100 °C for 5 min before loading into gel. For U2OS, NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) was used. For MEFs, NuPAGE Novex 3–8% Tris-Acetate Gel (Invitrogen) was used. Samples were run on gels for 2 hr before transfer from gel to a nitrocellulose membrane. Antibodies used to bind proteins on membrane were pAb rabbit BRIP1/FANCJ antibody (1/10000, Novus Biologicals NBP1-31883), pAb rabbit RTEL1 antibody (1/5000, Novus Biologicals NBP2-22360). For loading control, mAb mouse anti-β-actin antibody (1/5000, Abcam ab8226) was used with U2OS and mAb mouse anti-α-Tubulin (1/10000, Sigma Aldrich T6199) was used with MEFs. Secondary antibodies used were: pAb swine anti-rabbit immunoglobulins/HRP (1/10000, Dako P0217) and pAb goat anti-mouse immunoglobulins/HRP (1/5000, invitrogen A16078). Visualisation was performed by exposure onto photographic film for U2OS and visualisation using Amersham ImageQuant 800 for MEFs. Full uncropped scans are available in the source data file.