Hpa005848

Cells were lysed using (9 M urea, 50 mM Tris–HCL, pH 7.5, 150 mM β-mercaptoethanol) followed by sonication using a Soniprep 150 sonicator. Samples were resolved by SDS-PAGE and transferred to PVDF or nitrocellulose. Protein concentrations were determined by Bradford assays via spectrophotometry using a DeNovix DS-11 FX + spectrophotometer. Immunoblots were carried out using the indicated antibodies: α-Tubulin (Sigma, B-5-1-2; T5168, 1:100,000), BLM (Bethyl, A300-110A 1:2000), DNA2 (Abcam ab962488, 1:1000), EXD2 (Sigma, HPA005848, 1:1000), MCM2 (Abcam, ab4461, 1:10,000), MRE11 (Abcam, ab214, 1:1000), MUS81 (Abcam, ab14387 1:1000), POLD3 (Abnova, H00010714-M01, 1:500), RAD52 (28045-1-AP, Proteintech, 1:2000), SMARCAL1 (Santa Cruz, sc-376377 1:1000), SLX4 (University of Dundee, DU16029, 1:200), Vinculin (Thermo-Fisher, MA5-11690, 1:1000).

Cell lysis was carried out in urea buffer (9 M urea, 50 mM Tris HCL, pH 7.3, 150 mM β-mercaptoethanol) followed by sonication using a soniprep 150 (MSE) probe sonicator. In some instances, cells were lysed in SDS loading buffer (2% SDS, 10% (v/v) glycerol, 2% 2-Mercaptoethanol and 62.5 mM Tris-HCl, pH 6.8) followed by boiling for 10 min. Samples were resolved by SDS-PAGE and transferred to PVDF or nitrocellulose. Protein concentrations were determined by Bradford assay by spectrophotometry using a NanoDrop 2000 device (Thermo Scientific). Immunoblots were carried out using the indicated antibodies: α-Tubulin (Sigma, B-5-1-2; T5168, 1:100,000), BRCA1 (Millipore, OP-92, 1:1000), BRCA2 (Millipore, OP-95, 1:1000), EXD2 (Sigma, HPA005848, 1:1000), MCM2 (Abcam, ab4461, 1:10,000), MRE11 (Abcam, ab214, 1:1000), PCNA (Santa-Cruz, PC-10, 1:500), RECQ1 (Santa Cruz, sc-166388,1:1000) and SMARCAL1 (Santa Cruz, sc-376377 1:1000).