Western Blot Analysis of Protein Samples

OBSC sections (4-6) were pooled and lysed with Tris buffer and centrifuged at 21,000g to remove the nuclear fraction as we previously reported (48 (link)). Protein concentrations were measured using a Pierce BCA assay kit with the manufacturer’s instructions (ThermoFisher Scientific). Samples were diluted in RIPA and DTT containing reducing buffer (Pierce TM Cat. # 39000) to a final amount of 40 μg protein per well. Protein samples were separated on a 4–15% Ready Gel Tris-HCL gel (BioRad) and transferred onto PVDF membranes (BioRad). PVDF membranes were incubated at 4°C overnight with primary antibody. The following day, secondary antibody incubation (Li-Cor IRDye^® 680 or 800) was performed, and membranes visualized using the LiCor Odyssey™ imaging system. Values for proteins of interest were normalized to beta-actin expression for each sample.