Horseradish peroxidase hrp conjugated goat anti rabbit igg secondary antibody

The CVEC proteins were lysed in RIPA buffer containing protease inhibitors. Then, the protein concentrations were detected using the BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Next, the protein sample was electrophoresed by 8% SDS-PAGE and blocked with 5% bovine serum albumin and then was incubated with the primary antibody: anti-Bcl2 (Proteintech 60718-1-lg, Wuhan, Hubei, China), anti-Bax (Proteintech50599-2-lg, Wuhan, Hubei, China), anti-Caspase 3 (Cell Signaling Technology 9661s, Danvers, MA, United States), anti-Cytochrome C (Proteintech 10993-1-AP, Wuhan, Hubei, China), anti-p-AKT (Cell Signaling Technology 4060S, Danvers, MA, United States), anti-p-PI3K (Cell Signaling Technology 4228S, Danvers, MA, United States), and anti-GAPDH (Cell Signaling Technology 5174S, Danvers, MA, United States). Then, the primary antibody was washed out for 3 times and the membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Cell Signaling Technology 7074S/7076S, Danvers, United States) for 1.5 h at room temperature. The intensity of immune response was observed the with Image J software (1.48u, National Institutes of Health, United States).

Four matched pairs of WDTC tumor tissues and adjacent nontumor tissues were harvested and lysed in 50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mM sodium orthovanadate, 10 μM of a protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein were electrophoretically separated on a 9% SDS polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated at 4°C overnight with anti-human PDE5 rabbit monoclonal antibody (ab64179, 1 : 1000; Abcam Inc., Cambridge, USA). PDE5 expression was detected with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG secondary antibody (1 : 10,000; Cell Signaling Technology, USA). The immunoreactive bands were visualized with an ECL chemiluminescence system (Tanon, Beijing, China). Anti-β-actin mouse monoclonal antibody (1 : 1000; Cell Signaling Technology, USA) was used as a loading control.