Brain Protein Extraction and Western Blot

Total protein from brain tissues was extracted by using RIPA Lysis Buffer (P0013, Beyotime, Shanghai, China) with 1 mM PMSF following the manufacturer’s instructions. 30 g proteins were boiled at 100°C for each sample with protein loading buffer for 5 min, followed by separation in 10–12% SDS-PAGE electrophoresis and transferred onto PVDF membranes. Then, 5% lipid-free milk/TBST buffer were used to block the membranes at room temperature overnight, incubated with anti-p-ERK1 + anti-ERK2 (ab76299, 1:5000, Abcam), anti-CREB (ab32515, 1:1000, Abcam), anti-BNDF (ab108319, 1:1000, Abcam) and anti-GAPDH (ab8245, 1:5000, Abcam) primary antibodies for 2 h at 4°C overnight, respectively. After being incubation with secondary antibodies anti-mouse IgG (BA1051, 1:20,000, BOSTR) or anti-rabbit IgG (BA1054, 1:20,000, BOSTR,) for 1–2 h at room temperature, the immuno-complexes were finally detected by ECL after washing by TBST and analyzed using the Image-Pro Plus 6.0 software [30 (link)].