Peroxidase linked goat anti rabbit igg secondary antibody

Tissue samples of the upper genital tract were homogenized and lysed in ice-cold RIPA lysis buffer, then mixed with 2× SDS-PAGE sample buffer, boiled for 10 min, and then resolved by 10% SDS-PAGE. The proteins were then transferred onto polyvinylidene difluoride (Millipore, Billerica, MA, USA) membranes, blocked at 37°C for 60 min with 5% nonfat dry milk, and then reacted with properly diluted monoclonal antibodies (1:1000) including NF-κB p65, p-NF-κB p65, p-IκBα, Bα, BAX, BCL-2, JNK, and p-JNK. After washing, the membranes were incubated with peroxidase-linked goat anti-rabbit IgG secondary antibody (1:1000; Santa Cruz Biotechnology) at 37°C for 1 h. Protein bands were detected using horseradish peroxidase-conjugated goat anti-mouse IgG antibodies followed by an enhanced chemiluminescence reaction (Pierce Biotechnology, USA).

Total proteins extracts of each cell treatment group were resolved by 12% SDS-PAGE and transferred onto polyvinylidene difluoride (Millipore, Billerica, MA, USA) membranes. The membranes were blocked with Tris-buffered saline containing 0.3% Tween-20 (TBST) and 5% normal goat serum at 37°C for 60 min. Subsequent to blocking, the membranes were washed four times for 15 min with TBST at room temperature and then incubated with the following primary polyclonal antibodies: Rabbit anti-human EV71 (1:1,000; Millipore), rabbit anti-human CDK4, rabbit anti-human caspase-3, rabbit anti-human active caspase-3, rabbit anti-human Bcl-2, rabbit anti-human BAX and rabbit anti-GAPDH (1:1,000; all Cell Signaling Technology, Inc., Danvers, MA, USA). The membranes were washed four times for 15 min with TBST at room temperature. Following washing, the membranes were incubated at room temperature with peroxidase-linked goat anti-rabbit IgG secondary antibody (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h. Protein bands were visualized by autoradiography, using an enhanced chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA).