Anti mcm4

Each protein sample was mixed well with the same volume of 2X Laemmli’s buffer, boiled at 100°C for 3–5 min, and then loaded onto 10 or 12.5% SDS-PAGE gels. The proteins were then transferred onto nitrocellulose membrane. TBST containing 5% dry milk was used to block the membrane for 30 min, and the proteins on the membrane were probed with specific primary antibodies: Anti-WDR18 (anti-hIPI3; Proteintech, 15165-1-AP, 1:800), Anti-MCM3 (Santa Cruz, sc-9850, 1:1,500), Anti-MCM4 (Santa Cruz, sc-9840, 1:1,500), Anti-MCM6 (Santa Cruz, sc-9845, 1:1,500), Anti-ORC2 (Santa Cruz, sc-32734, 1:1,500), Anti-ORC3 (Santa Cruz, sc-21862, 1:2,000), Anti-ORC4 (Santa Cruz, sc-19725, 1:2,000), Anti-Histone (Santa Cruz, sc-11278, 1:2,000), anti-BM28 (Mcm2; Becton Dickinson, 610701, 1:1,500), anti-Mcm7 (Santa Cruz, sc-9966, 1:1,000), anti-Cdc6 (Santa Cruz, sc-9964, 1:1,500), anti-Orc6 (Santa Cruz, sc-32735, 1:1,500), anti-β-actin (Sigma, A2228, 1:1,000), or anti-HA (Roche, 12CA5, 1:20,000). The signals were visualized using the SuperSignal^™ reagent (Pierce) after secondary antibodies bound to the primary antibodies on the membrane.

Cancer cells were collected and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured by BCA assay. Samples in SDS sample buffer were heat-denatured at 95 °C for 5 min. The proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with primary antibodies including anti-MCM2 (sc-373702, 1:1000 dilution), anti-MCM3 (sc-390480, 1:1000 dilution), anti-MCM4 (sc-28317, 1:1000 dilution), anti-MCM6 (sc-393618, 1:1000 dilution), anti-AR (sc-7305, 1:1000 dilution), anti-SYP (sc-17750, 1:2000 dilution), anti-CD56 (sc-7326, 1:500 dilution), anti-cleaved PARP1 (sc-56196, 1:1000 dilution), anti-β-Actin (sc-47778, 1:2000 dilution), and anti-GAPDH (sc-47724, 1:2000 dilution) purchased from Santa Cruz Biotechnology. Secondary HRP conjugated antibody (PI31432, 1:6000 dilution) was purchased from Fisher Scientific. Western blot development and detection was performed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and IVIS Lumina optical imaging system (PerkinElmer). All the raw images of Western blot were summarized in Supplementary Fig. 4.