Anti actin rabbit polyclonal antibody

Cerebral tissues were sampled at 4, 12, 24 and 72 h after ischemia whereas cultured cells were collected at 24 h after OGD. Western blots were performed using protocols as previously described [17 (link)]. The following antibodies were used: rabbit anti-ASPP1 polyclonal antibody (1:200, Abbiotec, San Diego, CA, USA), rabbit anti-ASPP2 polyclonal antibody (1:200, Abbiotec), rabbit anti-iASPP polyclonal antibody (1:1000, Abcam, Hong Kong, China), rabbit anti-p53-upregulated mediator of apoptosis (Puma) (1:1000, Abcam), rabbit anti-Bax (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), cleaved caspase-3 (1:1000, Cell Signaling Technology Inc. Beverly, MA, USA) and rabbit anti-Actin polyclonal antibody (1:2000, Santa Cruz Biotechnology Inc.). Membranes were then incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2000, Santa Cruz Biotechnology Inc.). The blots were visualized by chemiluminescence (Millipore, Billerica, MA, USA). Protein levels were normalized to β-Actin as a loading control. The relative optical density of protein bands was measured after subtracting the film background.

Fibroblasts were chilled, washed twice with cold (4°C) PBS and scraped into the buffer. The cell were pelleted and solubilized in 50 μL of the cell lysis buffer (50 mM Tris-HCl, 2 mM CaCl₂, 0.1% Triton X-100) containing the serine protease inhibitors, aprotinin (19 μg/mL), benzamidine (1mM) and PMSF (1mM). The amount of protein in the cell lysates was quantitated by the method of Lowry [16 (link)].
Equal amounts of protein were resolved under denaturing conditions in a 7.0% polyacrylamide gel and transferred onto polyvinylidine fluoride membrane (Immobilon^™-P) in transfer buffer (25 mM Tris, 192mM glycine, 15% v/v methanol, pH 8.5) at 30 volts overnight or at 100 volts for 1 hour. The blot was first incubated in blocking buffer (20mM Tris, 150mM NaCl, 2mM CaCl₂, 1% gelatin, 0.05% TWEEN-20, pH 7.4) overnight at room temperature and then probed with anti-ABCA1 rabbit polyclonal antibody (5 μg/mL, Novus Biologicals) and peroxidase-conjugated goat-anti-rabbit secondary antibody (Jackson Immuno Research Laboratories, Inc.). The Supersignal West Pico chemiluminescent substrate (Pierce) was used to detect the protein bands. The membrane was stripped and reprobed with anti-actin rabbit polyclonal antibody (1.5 μg/mL, Santa Cruz Biotechnology) and goat-anti-rabbit secondary antibody. Results of Western Blot analysis were quantitated using the ImageJ software available at the NIH website.