β actin antibody

THZ1 (#M5228) was purchased from AbMole BioScience, Inc. (Houston, TX, USA). Antibodies against various proteins for Western blot analyses, such as CDK7, RNAPII, RNAPII pS5, RNAPII pS7, cleaved poly ADP ribose polymerase (PARP), cleaved caspase-3, cleaved caspase-7, VEGFR2, CD31, and VEGF, were obtained from Cell Signaling Technology (Danvers, MA, USA). The β-actin antibody was purchased from GeneTex (Irvine, CA, USA), and the α–tubulin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), Merck Millipore (Billerica, MA, USA), and Invitrogen (Carlsbad, CA, USA).

Samples of the left hippocampus or cortex were homogenized in PBS containing complete protease inhibitors in a glass vesicle; the resulting homogenate was centrifuged at 12,000 rpm at 4°C for 20 min; and the protein concentrations of the supernatant thus obtained determined with the BCA protein assay kit. Proteins (30 μg) were subsequently separated by 8-10% SDS-PAGE and then blotted onto polyvinylidene difluoride (PVDF) membranes with a transfer unit (Bio-Rad Inc.). For the relative quantification of the proteins, these membranes were thereafter incubated with antibodies against α7 (1:1000), α4 (1:1500) and β2 (1:1000), cleaved caspase-3 (1:1000, Gene Tex, USA), Bcl-2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Snap-25 (1:1000, Gene Tex, USA), Syn (1:1000, Gene Tex, USA) or β-actin antibody (1:5000, Gene Tex, USA) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Gene Tex, USA) for 60 min. Finally, these membranes were incubated in ECL Plus (Pierce, Thermo Fisher Scientific) reagent and the signals thus obtained visualized by exposure to hyper-performance chemiluminescence film for 30 sec to 5 min.