F 11 sc 374598

Acute exposure of cells to MMC (Sigma) occurred in regular growth medium at 37°C for 2 h and at the concentrations, as indicated. The medium was removed, and monolayers were washed twice in warm PBS before cells were incubated in regular growth medium at 37°C until fixation. For chronic exposure to MMC, cells were plated in regular growth medium containing MMC and kept at 37°C for 12 d, at which time the cells were fixed and stained with crystal violet to visualize colonies. Exposure to γ-rays and Western blot analyses followed our standard protocols (Parplys et al., 2014 (link); Parplys et al., 2015 (link); Wiese et al., 2006 (link); Zhao et al., 2015 (link)). The primary antibodies that were used are α-RAD51AP1 (NB100-1129; Novus; 1:5,000; and our own α-RAD51AP1 antibody, as previously described in Dray et al., 2010 (link)), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-NUCKS1 (Østvold et al., 2001 (link)), α-RAD51 (Ab-1; EMD Millipore; 1:4,000), α-PARP1 (ab6079; Abcam; 1:5,000), α-H3 (ab1791; Abcam; 1:10,000), α-XRCC4 (AHP387; AbDSerotec; 1:1,000), and α-FLAG (F3165; Sigma; 1:1,000). HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; 1:10,000) were used as secondary antibodies. Western blot signals were quantified using Image Lab software version 5.2.1 (BioRad).