Nupage novex 4 12 gels

The dissected CPu and NAc (Famous et al., 2008 (link); Jin et al., 2019 (link)) were homogenized in a radioimmunoprecipitation assay buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% sodium deoxycholate, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM β-glycerophosphate, 1 μg/ml leupeptin, and a protease/phosphatase inhibitor cocktail (Thermo Scientific, Rochester, NY). Homogenates were centrifuged at 800 g (10 min, 4°C). Supernatants were collected and protein concentrations were determined. Western blot assays were conducted as described previously (Jin et al., 2013 (link)). Briefly, sodium dodecyl sulfate NuPAGE Novex 4–12% gels (Invitrogen, Carlsbad, CA) were used for separation of proteins. Separated proteins on gels were then transferred to polyvinylidene fluoride membranes. Membranes were blocked and washed. They were incubated with a primary rabbit antibody (see below) overnight at 4°C. An anti-rabbit secondary antibody was incubated with membranes. We used an enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Piscataway, NJ) to visualize proteins. Optical density of immunoblot bands was measured using NIH ImageJ analysis software. All optical density values were normalized to a loading control protein (actin).