Anti mcm7 antibody

Anti-TRAIP, -GFP and -γH2AX antibodies were previously described (34 (link),35 (link)). Other antibodies were purchased as the followings: anti-Flag-HRP (Sigma, A8592), anti-Myc-HRP antibody (Roche, 11814150001), anti-GFP (Clontech, 632380), anti-β-actin antibody (Sigma, A5441), anti-α-tubulin antibody (Millopore, 05–829), anti-ZNF212 antibody (Altas antibodies, HPA049807), anti-TRAIP antibody (30 (link)), anti-RAD51 antibody (Abcam, ab3801), anti-RPA2 antibody (Bethyl laboratories, A300-244A), anti-53BP1 (Cell signaling technology, #4937), anti-BRCA1 antibody (Santa cruz, SC-6954), anti-FANCD2 antibody (Novus Biologicals, NB100-182), anti-Histone 3 antibody (Santa cruz, sc-8654), anti-NEIL3 antibody (Proteintch, 11621-1-AP) and anti-MCM7 antibody (Santa cruz, sc-9966).

For EdU FACS, cells were treated with 10 μM EdU for 10 min, trypsinized, washed with PBS, and fixed with cold 70% ethanol on ice for 30 min to overnight. Cells were washed with PBS, and EdU staining was performed by using the EdU Click-iT kit (Thermofisher, # C10632), according to the manufacturer’s instructions. For DNA staining, we used 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) or FxCycle™ PI/RNase Staining Solution (Thermofisher, #F10797).
Chromatin association of MCM was assessed essentially as described^{46 (link)}: after trypsinization and PBS wash, cells were extracted with CSK buffer (10 mM PIPES pH 7.0, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl₂, 0.5% Triton X-100, protease inhibitors (Pierce #A32953), fixed with 4% paraformaldehyde, and blocked with 5% BSA followed by immunostaining with anti-MCM7 antibody (Santa Cruz, #sc-9966) at 1:200 dilution. 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) was used for DNA staining.
Flow cytometry was performed on an FACSCalibur flow cytometer, and data were analyzed by using FCSalyzer software. GraphPad Prism 9 was used for statistical analyses.