Gapdh sc 48167

Ischemic brain sections were homogenized in Tissue Lysate Buffer (Beyotime, Shanghai, China), and phases left to separate on ice for 30 minutes. The protein phase was then centrifuged at 8,000 × g for 20 minutes, and the supernatant removed. Protein concentration was measured with a BCA kit (Beyotime, Shanghai, China). For each sample, 15 μg total protein was mixed with 4 μL 5 × loading buffer and denatured by boiling for 5 minutes. Subsequently, specimens were separated by electrophoresis on 10% SDS-PAGE gels (Beyotime, Shanghai, China) at 80 V for 40 minutes and at 110 V for 90 minutes, and then transferred to polyvinylidene difluoride membranes (Beyotime, Shanghai, China) at 200 mA for 60 minutes. After blocking for 2 hours, membranes were washed with western solution (Beyotime) and incubated with goat anti-rat polyclonal antibody (SMAD2/3 sc-6032, pSMAD2/3 sc-11769, Bcl-2 sc-16323, caspase-3 sc-1225, cleavage casp3 sc-22139, GAPDH sc-48167) 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, and with horseradish peroxidase conjugated rabbit anti-goat polyclonal antibody (sc-2768) 1:1,000 (Santa Cruz Biotechnology) for 2 hours. Antibody binding was visualized with the ECL method, and results are expressed as the gray value ratio of target protein to GAPDH.