Western Blotting of Uterine and Oviduct Proteins

Protein lysates (40 µg per lane) from uteri or oviducts were loaded on a 4–15% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA), separated in 1X Tris-Glycine Buffer (Bio-Rad), and transferred to PVDF membranes via a wet electro-blotting system (Bio-Rad), all according to the manufacturer’s directions [20 (link)]. PVDF membranes were blocked for 1 hour in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T, Bio-Rad), then incubated overnight at 4 °C with antibodies listed in Supplementary Table 2 in 5% BSA in TBS-T. Blots were then probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1:5000, Cell Signaling Technology, Danvers, MA, USA) in 5% BSA in TBS-T for 1 hr at room temperature. Signal was detected with the Pierce^™ ECL Western Blotting Substrate (Millipore, Billerica, MA, USA), and blot images were collected with a Bio-Rad ChemiDoc imaging system.