Hybond p pvdf

Cell extracts were lysed on ice with RIPA buffer (phosphate‐buffered saline, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing 0.1 mg/mL phenylmethyl sulfonyl fluoride, 30 mg/mL aprotinin, and 1 mmol/L sodium orthovanadate, scraped for 20 seconds, and centrifuged at 14 000 g for 10 minutes at 4°C. Protein concentration in the cell lysates was measured using the Bradford method. Denatured protein (30 µg per well) was resolved in 10% SDS polyacrylamide gel electrophoresis (SDS‐PAGE) gels according to standard protocols. Protein was then transferred onto polyvinylidene difluoride membranes (Hybond‐P PVDF; Amersham Biosciences), which were blocked for 2 hours at room temperature in Blotto (5% milk in Tris‐buffered saline). Membranes were incubated with anti‐RXFP3 antibody (1:100 dilution; Santa Cruz Biotechnology, Inc) in Blotto overnight at 4°C and washed three times for 10 minutes per wash with Tris‐buffered saline/1% Tween. Subsequent incubation with horseradish peroxidase‐conjugated (HRP‐conjugated) antibodies was performed for 1 hour at room temperature in Blotto, and additional washes were performed as needed. Following enhanced chemiluminescence detection (Amersham Biosciences), membranes were exposed to X‐ray film (Fujifilm). Tissues from rat cerebral cortex were used as positive controls.

For immunoblot analyses, 40 μg of protein lysates per sample was denatured in 4x SDS-PAGE reducing sample buffer and subjected to SDS-PAGE on 10% acrylamide/bisacrylamide gels. Separated proteins were transferred to nitrocellulose membrane (Hybond-P PVDF, Amersham Bioscience). Residual binding sites on the membrane were blocked with 5% (w/v in TBST) nonfat milk overnight at 4°C. Membranes were then probed with specific primary antibodies, anti-NF-κB p65 rabbit monoclonal antibody (Santa Cruz Biotechnology) (1 : 400) and anti-Lamin B mouse monoclonal antibody (Santa Cruz Biotechnology) (1 : 700), followed by peroxidase-conjugated secondary antibody, anti-rabbit Ig (Cell Signaling Technology) (1 : 5000) and anti-mouse Ig (Cell signaling Technology) (1 : 5000), and visualized with an ECL plus detection system (Amersham Biosciences). The equivalent loading of proteins in each well was confirmed by Ponceau staining and Lamin B control.

Caspases were analyzed by a Western blot analysis. For immunoblot analyses, 40 µg of protein lysates per sample were denatured in SDS-PAGE sample buffer (Tris-HCl 260 mM, pH 8.0, 40% (v/v) glycerol, 9.2% (w/v) SDS, 0.04% bromophenol blue and 2-mercaptoethanol as a reducing agent) and subjected to SDS-PAGE on 5% acrylamide/bisacrylamide gels. Separated proteins were transferred to nitrocellulose membrane (Hybond-P PVDF, Amersham Biosciences). Residual binding sites on the membrane were blocked by incubation in TBST (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) with 5% (w/v). Membranes were then probed with a specific primary antibody, Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA) (1:200). This was followed by a peroxidase-conjugated secondary antibody, HRP labeled mouse antirabbit Ig (Cell Signaling Technology, Danvers, MA, USA) (1:10000), and visualized with an ECL Plus detection system (Amersham Biosciences). The equivalent loading of proteins in each well was confirmed by Ponceau staining [30 (link)].