Ev71 vp1

The cells were lysed in the M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) containing halt protease inhibitor single-use cocktail (Thermo). The protein concentration was determined by the BCA reagents (Thermo). About 15 μg proteins were denatured and applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis products were transferred to a polyvinyl idenefluoride (PVDF) film and PVDF membranes were then incubated at room temperature with specific primary antibody. After a standard washing, membranes were incubated with horse radish peroxidase (HRP)-labeled secondary antibody. The assay developed using a chemiluminescent substrate. The primary antibodies used in this study included antibodies against β-actin, p-p44/p42 MAPK, p44/p42 MAPK, p-MEK, MEK, p-AKT, AKT, p-JNK, JNK, PI3KIII, SQSTM1/P62, LC3B, Beclin-1 (Cell Signaling Technology), and EV71-VP1 (Abnova). The goat anti-rabbit and anti-mouse HRP-labeled antibodies were obtained from Cell Signaling Technology.

Cells were washed with cold PBS and treated with RIPA buffer (Thermo, Rockford, IL) containing protease inhibitor cocktail (Roche) and 1% phenylmethylsulfonyl fluoride PMSF (Beyotime, Shanghai, China). Proteins were quantified by Bradford assay and separated in 12% SDS-PAGE prior to transferring onto polyvinylidene difluoride (PVDF) membrane. Protein bands were visualized by enhanced chemiluminescence technique using SuperSignal West Pico chemiluminescent substrate (Thermo, USA). Densitometric quantification was performed using ImageJ v1.48 and the relative band intensity for each protein of interest was normalized against β-actin. The primary antibodies used in this study included antibodies against β-actin (Proteintech, Wuhan, China), caspase-1 (Cell Signaling), EV71 VP1(Abnova), IL-1β (Proteintech), IL-18 (Proteintech), and NLRP3 (Biovector, Shanghai, China). The goat anti-rabbit or anti-mouse horse radish peroxidase (HRP)-labeled antibodies were obtained from Proteintech.