Protein extraction, chromatin fractionation, and western blotting

For preparation of whole-cell extracts, cells were lysed in RIPA (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM NaF, 1% NP-40, 0.1% SDS, 0.4 mM EDTA, 0.5% sodium deoxycholate, 10% glycerol) buffer for 10 min and then centrifuged at 16,000 g for 10 min. Cleared lysates were collected, mixed with SDS loading buffer and boiled before fractionation by SDS-PAGE and analyses by western blot. Extracts were prepared⁷² by lysis in Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100 and protease inhibitors). Insoluble nuclear proteins were isolated by centrifugation and chromatin-bound proteins were subsequently released by sonication. The remaining insoluble factors were cleared by centrifugation before fractionation by SDS-PAGE and western blot analyses. Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: rabbit anti-MCM10 (Bethyl A300-131A; 1:500), rabbit anti-MCM10 (Novus, H00055388-D01P, 1:500), mouse anti-CDC45 (Santa Cruz G12, SC55568; 1:500), mouse anti-MCM4 (Santa Cruz G7, SC28317; 1:500), mouse anti-MUS81 (Abcam ab14387; 1:500), mouse anti-PCNA (Abcam Ab29; 1:3,000), rabbit anti-Ubiquityl-PCNA (Lys164) (Cell Signaling Technology mAb13439, D5C7P; 1:1,000), rabbit anti-RPA32 (S4/8) (Bethyl A300-245A; 1:2000), mouse anti-GAPDH (GeneTex GTX627408; 1:5,000), mouse anti-α-Tubulin (Millipore T9026, clone DM1A; 1:10,000), rabbit anti-Lamin B1 (Proteintech #12987; 1:3,000). Secondary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) at 1:10,000 dilutions, including goat anti-mouse HRP conjugate (Jackson Laboratories 115-035-003), goat anti-rabbit HRP conjugate (Jackson Laboratories 111-035-144), goat anti-mouse HRP conjugate (BioRad, 1706516), donkey anti-rabbit HRP conjugate (Amersham NA9340). Detection was performed using WesternBright Quantum detection kit (K-12042-D20). Quantification was performed using FIJI version 2.1.0/153.c and Microsoft Excel version 16.44. Image preparation was performed using Adobe Photoshop version 21.2.1.