Mouse anti human gapdh antibody

At 48 h after transfection, cells were lysed with RIPA buffer (Invitrogen) and western immunoblotting was performed according to the standard process. The major antibodies applied to the analysis were anti-human and mouse VEGF-A antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human CD31 antibody (1:500, Abcam, Cambridge, MA, USA), rabbit anti-Ki-67 (1:500, Santa Cruz Biotechnology), anti-MMP-9 mouse antibody (1:700, Abcam) and mouse anti-human GAPDH antibody (1:1000, Santa Cruz Biotechnology) as a loading regulator.

Tissues (100 mg; homogenized tissues by grinding in liquid nitrogen) and cultured cells (1.5×10⁶ cells) were lysed with RIPA buffer (Beyotime Institute of Biotechnology) to isolate total protein. The concentration of total protein was measured with the Bicinchoninic Acid Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein were separated by 10% SDS-PAGE, electrotransferred onto PVDF membranes, and blocked at room temperature for 2 h with 5% fat-free milk diluted with Tris-buffered saline containing 0.1% Tween-20 (TBST). Subsequently, the membranes were incubated at 4°C overnight with the following primary antibodies: Mouse anti-human CCNB1 antibody (cat. no. sc-7393; 1:1,000; Santa Cruz Biotechnology, Inc.) and mouse anti-human GAPDH antibody (cat. no. sc-69778; 1:1,000; Santa Cruz Biotechnology, Inc.). Following three rinses with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (cat. no. 516102; 1:5,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The Amersham ECL Western Blotting Detection kit (GE Healthcare Life Sciences) was used for protein signal detection. GAPDH served as the loading control and for normalization of protein expression. Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.) was used for densitometric analysis.