Anti b cell lymphoma 2 bcl 2

Protein was extracted from total brains (n = 4 per experimental group, at 72 h after the transplantation of BMSCs) and cultured cortical neurons (at 4 h after OGD insult). A 50 µg protein aliquot was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and blotted onto polyvinylidene difluoride membrane (Millipore). After blocking with 5% fat-free milk at RT for 2 h, blots were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti-HGF (1:500), anti-p-ERK (1:500), anti-B-cell lymphoma-2 (Bcl-2, 1:500), and anti-β-actin (1:500) (all obtained from Santa Cruz Biotechnology). After washing, the blots were incubated with secondary antibodies (1:200, Santa Cruz Biotechnology) at RT for 2 h. Finally, membranes were washed and specific bands were detected with an enhanced chemiluminescence system (ECL, Pierce, Rockford, IL, USA). Data from protein densitometry were quantitatively analyzed using Labworks Analysis Software (Upland, CA, USA) and normalized to levels of the housekeeping protein, β-actin.