Western Blot Analysis of ORC6 Protein

We followed the methods of Qiufeng Pan et al. 202016 (link). Cells and tissues were lysed in a protein lysis system including PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China), protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA), and RIPA buffer (Beyotime Institute of Biotechnology). Protein concentration was measured by the BCA kit (Beyotime Institute of Biotechnology). A total of 30μg proteins were presented and separated in 10% SDS-PAGE. Then, the gel system was transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA) for 90 mins at 90 V, after which the PVDF membrane was blocked in 5% nonfat milk dissolved in PBS for 1 h and then incubated with antibodies against ORC6 (1:1,000; A5426; Abclonal Biotec Co., Ltd) and β-actin (1:3,000; ab8226; Abcam Co., Ltd) at 4°C overnight. The next day, the membrane was washed and incubated with secondary antibodies (1:3,000; GB23303; Servicebio, Inc.) for 2 h at room temperature. Finally, the proteins were visualized with ChemiDoc-XRS⁺ (Bio-Rad Laboratories, Inc., Hercules, CA, USA).