Western Blotting to Assess MCM10 Expression

The western blotting assay was performed based on that of our previous publications [45 (link), 46 (link)] to evaluate the endogenous MCM10 expression and the efficiency of MCM10 knockdown in J82 and TCCSUP cell lines. Cell lysates containing 25 μg protein were separated by 4-12% gradient NuPAGE gel (Invitrogen, Carlsbad, CA), transferred onto PVDF membranes (Amersham, Biosciences, Buckinghamshire, UK). After blocking with 5% skimmed milk in TBST buffer at room temperature for 1 h, the membranes were then probed with antibodies at 4°C overnight against MCM10 (1:1000, H-41, Santa Cruz), and GAPDH as a loading control (6C5, 1:10,000, Millipore, Beverly, MA). After incubation with the secondary antibody at room temperature for 1.5 h, proteins were visualized by the chemiluminescence system (Amersham Biosciences).