Anti pcreb antibody

The cells were incubated for 12 h in the presence or absence of 2.5 μg/ml of DMGF. Then, the cells were collected and lysed in ice-cold RIPA lysis buffer. The protein concentration of the cell lysate was estimated with the Bradford protein assay using BSA as the standard. Total proteins (50−60 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate buffered saline with Tween 20 (0.05% v/v Tween-20 in PBS, pH 7.2) for 1 h. The membranes were then incubated with anti-CREB antibody (1:1,000; GeneTex, Irvine, CA, USA) and anti-pCREB antibody (1:1000; Abcam, Cambridge, CA, USA) at 4°C overnight, followed by incubation with a horseradish peroxide-linked secondary antibody (1:10000; GeneTex, Irvine, CA, USA). The protein bands were visualized using the ChemiLucent ECL Detection System (Millipore, Billerica, MA, USA) and the Biospectrum AC Imaging System (UVP, Upland, CA, USA). The intensities of the chemiluminescence signal were quantified using the UVP VisionWorks LS Image Acquisition and Analysis Software (UVP, Upland, CA, USA).

To detect protein levels, mouse brains were harvested 6 hours after the onset of stroke and then homogenized in RIPA lysis buffer (Boston Bioproducts). The concentration of protein in each sample was determined with a BCA assay (Thermo Scientific). The proteins were separated using precast gels (7.5%, 10%, 12% or 4-15%, Bio-Rad) and were then transferred to polyvinylidene difluoride membranes (Bio-Rad). After the transfer and blocking steps, the blots were incubated overnight at 4°C with primary antibodies (anti-Collagen IV antibody, 1:500, Abcam; anti-p-CREB antibody, 1:1000, Abcam; or anti-BCL-2 antibody, 1:1000, Cell Signaling Technology) diluted in Tris-buffered saline containing 0.1% Tween-20 and 4% bovine serum albumin or 5% fat free milk. The appropriate secondary antibodies (anti-rabbit IgG, 1:5000, Cell Signaling Technology; or anti-mouse IgG, 1:5000, Vector) were diluted in the same blocking buffer, and an electrochemiluminescence detection kit (Thermo Scientific) was used for signal detection. β-actin (primary antibody 1:5000; Sigma) was used as a loading control.