Dorsal hippocampus was homogenized in RIPA buffer (Boston BioProducts, BP-115) with protease and phosphatase inhibitors (ThermoScientific, 1861281) using a dounce homogenizer. BAF53b homozygous knockout brain tissue was collected from P2 pups to verify antibody specificity. Protein lysates were quantified using a modified Bradford assay (BioRad, 500-0006). 10ug of total protein lysate were loaded per lane on a 4-12% Bis Tris gel (Novex) and run for 50min at 200V. Blots were then transferred overnight at 15V at 4C on to 0.45um nitrocellulose membranes (Novex). Total protein transfer was verified using PonseauS staining. Membranes were blocked in StartingBlock T20 (ThermoScientific, 37543) for 30min and then probed with rabbit anti-BAF53b (1:1,000; generous gift from G.R. Crabtree). The membranes were washed (3×10min in 0.1MPBS with .05%Tween20) and probed with donkey anti-rabbit-HRP (1:10,000; Jackson ImmunoResearch, 711-036-152). The membranes were washed and developed using Pierce SuperSignal West Pico Chemiluminescent Substrate (Pierce, 34077). Multiple film exposures were used to verify linearity. Blots were washed and then reprobed with rabbit anti-GAPDH (1:1,000; Santa Cruz Biotechnology, SC25778). Relative densities were taken from scanned film using ImageJ (NIH). All values were normalized to GAPDH expression levels.
