Proteins were transferred onto a nitrocellulose membrane and blocked in 5% milk in PBST for 30 min at 22°C. Membranes were reacted for 2–4 h at 22°C or overnight at 4°C with primary antibodies diluted in PBST. Primary antibodies used were as follows: rabbit anti-acSMC3 (1:1,000, MBL), rabbit anti-SMC3 (1:1,000, A300-060A; Bethyl Laboratories), mouse anti-SMC1β (1:2, hybridoma supernatant), guinea pig antimouse ESCO2 (1:500, [Whelan et al, 2011 (link)]), and mouse anti-GAPDH (1:200, sc-32233; Santa Cruz). Membranes were washed three times in PBST, and HRP-conjugated secondary antibodies were added for 1 h at 22°C in PBST. Secondary antibodies were all diluted 1:5,000 and were used as follows: antirabbit IgG HRP (18-8816-31; eBioscience), antimouse IgG HRP (115-035-003; Dianova), and anti–guinea pig IgG HRP (106-035-003; Dianova). A pre-stained protein ladder (#26619; Thermo Fisher Scientific) was loaded onto each gel to determine the molecular weight of the detected bands. Blots were washed three times in PBST and developed using chemiluminescent HRP substrate (WBKLS0100; Millipore) and imaged on a Kodak ImageStation 2000MM.
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