Anti creb antibody

Cells were collected and lysed in ice-cold RIPA lysis buffer. Sixty micrograms of protein was electrophoresed by SDS-PAGE using 10% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in phosphate-buffered saline with 0.05% Tween 20 for 1 h. The membranes were probed using anti-NF-κB antibody (1:200; Santa Cruz, Shanghai, CN), anti-CREB antibody (1:500; GeneTex, CA, USA), anti-HIF-1α antibody (1:500; GeneTex, CA, USA) or anti-β-actin antibody (GeneTex, CA, USA) followed by anti-mouse or anti-rabbit secondary antibodies (1:10,000; GeneTex, CA, USA). The protein bands were developed using the ChemiLucent ECL Detection System (Millipore, MA) and were visualized using the Biospectrum AC Imaging System (UVP, CA, USA).

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).