Ev71 vp1

The mouse polyclonal antibody against EV71 VP1 (~36 kDa, Abcam), the mouse monoclonal antibodies against PTEN (~54 kDa, Cell Signaling) and β-actin (~42 kDa, Abcam), the rabbit monoclonal antibodies against Akt (~60 kDa, Cell Signaling) and phosphor-Akt (~60 kDa, Cell Signaling), and the goat anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase (HRP). In addition, the mouse polyclonal antibody against EV71 VP1 (Abcam) and the goat anti-mouse Alexa Fluor 594 secondary antibody (Invitrogen) were used for the immunofluorescence assay in this study.

Cells were collected and lysed using RIPA lysis buffer (Santa Cruz, CA, USA) on ice for 20 min and then centrifuged at 12 000 g for 10 min at 4 °C. Total protein concentrations were determined using the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins were analyzed by SDS/PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Nonspecific antibody binding was blocked by Odyssey Blocking buffer (LI‐COR Biosciences, Lincoln, NE, USA), and the membranes were incubated with primary antibodies specific for SCARB2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), EV71 VP1 (Abcam, Cambridge, UK), or GAPDH (Santa Cruz Biotechnology) for 4 h at room temperature (RT). After five washes with PBS‐0.1% Tween‐20 (PBS‐T buffer), the membranes were incubated with IRDye 800 goat anti‐mouse IgG or IRDye 680 donkey anti‐rabbit IgG (LI‐COR Biosciences) at 1 : 10 000 dilution for 1 h and visualized using Li‐COR Odyssey Infrared Imager (LI‐COR Biosciences). Bands were quantified by densitometric analysis using odyssey software.