2.5. Western blot analysis

Hippocampi and whole brains from mice (two pooled hippocampus and one whole brain per age group) of both sexes at P0, P7, P14, and P21 were dissected for Western blot analysis of the temporal expression (six animals for hippocampus and three animals for whole brain per age‐point). Cortex, hippocampus, striatum, and cerebellum from four P14 mice (two to three pooled brain areas per sample, 8–12 animals) of both sexes were prepared for Western blot analysis of the spatial expression. Results from at least three independent cultures of N2a cells were collected for each experiment. Brain tissues or N2a cells transfected with plasmids were harvested in RIPA buffer containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP‐40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta‐glycerophosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, 1 mM PMSF, and a phosphatase and protease inhibitor cocktail (Millipore Sigma). Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). About 50 µg of total protein from N2a cell lysates was used to determine the knockdown efficiency of tomosyn shRNAs and to validate tomosyn domain mutants. Ten micrograms of total protein from different brain regions was used to examine tomosyn expression. Protein samples were run on 8% SDS‐PAGE gels and transferred to a PVDF membrane. After blocking the membrane with 5% skim milk in TBST (0.1% Tween 20 in TBS) for 1 hr at room temperature, the membrane was immunoblotted with the following primary antibodies: rabbit anti‐tomosyn (Synaptic Systems), mouse anti‐β‐Actin (Sigma‐Aldrich), mouse anti‐syntaxin‐1 (Synaptic Systems), mouse anti‐syntaxin‐4 (Synaptic Systems), mouse anti‐PSD‐95 (UC Davis/NIH NeuroMab Facility), rabbit anti‐RFP (Thermo Fisher Scientific), and rabbit anti‐GAPDH (Cell Signaling Technology). The membrane was washed three times with TBST for 10 min and incubated with the HRP‐conjugated secondary antibodies for 60 min at room temperature. The membrane was then washed five times with TBST for 10 min at room temperature. The chemiluminescent reaction was induced by incubating the membrane with Clarity™ Western ECL substrate (Bio‐Rad). The chemiluminescent blots were then imaged with the ChemiDoc™ Touch Imaging System (Bio‐Rad). The densitometry of the protein signals was analyzed using Image Lab™ software (Bio‐Rad).