Extraction and Analysis of Cellular Proteins

Total cellular proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) containing halt protease inhibitor single-use cocktail (Thermo). The extracted total proteins were denatured by adding 5× sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Thermo), followed by boiling for 5 min at 100°C. Approximately 15 μg proteins was applied for SDS-PAGE (Wang et al., 2017 (link)). The primary antibodies used in this study included antibodies against β-actin, p-JNK, JNK, SQSTM1/P62, LC3B (Cell Signaling Technology, Beverly, MA, USA), CVA16 (Millipore, MA, USA) and EV71 VP1 (Abnova, Taipei, China). The goat anti-rabbit and anti-mouse HRP-labeled antibodies were obtained from Cell Signaling Technology.