Anti gadph

Protein lysates were separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with anti-IκB kinase, anti-GADPH, anti-BCL2, anti-BAX, anti-p21, or anti-β-ACTIN antibodies (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C before subsequent incubation with secondary antibodies conjugated with horseradish peroxidase for 1 h at 37 °C. Proteins were visualized using enhanced chemiluminescence reagent.

RD cells or 293T cells infected with RNA transcripts or plasmids were cultured at 37°C with 5% CO₂ for different amounts of time and then lysed in a SDS–PAGE loading dye, followed by heating at 95°C for 10 min before separation by SDS-PAGE. Cells infected with wild type EV71 were used as positive controls and uninfected cells were used as negative controls. Total proteins on gels were transferred to nitrocellulose membranes at 250 mA for 2 h in transfer buffer (96 mM glycine, 12.5 mM Tris and 10% methanol). The membranes were blocked in 5% milk soluted in PBS-0.1% Tween 20 (PBST) at 37°C for 1 h, followed by incubation with indicated primary antibodies in PBST over night at 4°C. The membranes were washed three times in PBST and then incubated at 37°C for 1 h in secondary antibody diluted 1:10000 in PBST. The membranes were washed three times for 10 min and then detected by chemiluminescence.
Antibodies against IRF7, NF-κB, HRP Conjugated Goat anti Rabbit IgG H&L and Goat Anti-Mouse IgG H&L were purchased from abcam (UK). Anti-GADPH was purchased from Cell Signaling Technology (Danvers, MA). Anti-EV71 3C and anti-EV71 3D antibodies were purchased from GeneTex (UK).