Gapdh specific monoclonal antibody

Protein assays were performed according to the Bradford method using a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Denatured proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Hybond membranes (Amersham, Germany). The membranes were blocked for 1 hour in 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST; 10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20). Immunoblotting was performed with the following primary antibodies against GSK-3β, CDK1, CDK2, matrix metalloproteinase-9 (MMP9), cyclin D1, and Bcl-xL (1:500, Proteintech; Proteintech Group, Chicago, IL, USA). The membranes were incubated overnight at 4°C, rinsed with TBST, and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G antibodies (1:5,000; Dako Denmark A/S, Glostrup, Denmark). After applying ECL Plus detection reagents (Santa Cruz Biotechnology Inc., Dallas, TX, USA), protein bands were visualized using X-ray film (Fujifilm, Tokyo, Japan). The immunoblots were washed with Western blot stripping buffer (pH 2–3; Nacalai, Tokyo, Japan) and were probed using a GAPDH-specific monoclonal antibody (1:2,000; Santa Cruz Biotechnology Inc.).