Ecl plus western blotting detection system

Brain tissue was homogenized using a radioimmunoprecipitation assay. Total protein was determined using a bicinchoninic assay. A total of 50 µg protein was subjected to 8–12% SDS-PAGE analysis and transferred to nitrocellulose membranes. Membranes were blocked using 5% skimmed milk powder in TBS with 0.1% Tween-20 for 1 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: nNOS (cat. no. c-17825, 1:1,000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), β-amyloid (Aβ; cat. no. 8243, 1:2,000, Cell Signaling Technology, Inc.), amyloid precursor protein (APP; cat. no. sc-374527, 1:5,000, Santa Cruz Biotechnology, Inc.) and GAPDH (cat. no. sc-51631, 1:5,000, Santa Cruz Biotechnology, Inc.). Following this, membranes were incubated at 37°C for 1 h with corresponding horseradish peroxidase-conjugated secondary antibodies (cat. no. sc-2004, 1:2,000; Santa Cruz Biotechnology, Inc.). Membranes were visualized using the ECL Plus western blotting detection system (Beyotime Institute of Biotechnology), developed on a C-DiGit Blot Scanner (LI-COR Biosciences, Lincoln, NE, USA) and quantified using Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Membrane and cytosolic proteins were isolated with a Membrane and Cytosol Extraction Kit (Beyotime, China). Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, China). Equal amounts of proteins were separated by 10% SDS gel electrophoresis under denaturing and nonreducing conditions, and then transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline and 1‰ Tween 20 (TBST) at room temperature for 1 h, and then incubated with primary antibody at 4 °C overnight. After washing in TBST, the membrane was incubated with the secondary antibody. Membranes were exposed to enhanced chemiluminescent reagents (ECL Plus Western Blotting Detection System, Beyotime, China). Images were captured and analyzed using an ImageQuant LAS 4000 instrument.