Protease inhibitor cocktail

Tissues and cells and were harvested and lysed by RIPA (CST, USA) added with 1× protease inhibitor cocktail (Beyotime). Protein concentration was evaluated by a BCA quantification method. Subsequently, 35 μg extracted samples were separated on SDS-PAGE and transferred onto PVDF membranes. The blot was probed using specific primary antibodies (YAP (Abcam, USA), and β-actin (Abcam, USA)) at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 hour. The blots were washed with TBST for three times, followed by visualization via a Millipore ECL reagent (Millipore, USA) under a Gel imaging system (BD Biosciences, USA).

Whole-cell protein lysates were solubilized in 100-200 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) supplemented with a 1% protease inhibitor cocktail (Beyotime, China) for 20 min on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). The proteins (30 μg) were denatured and separated on an 8%-15% discontinuous SDS-polyacrylamide gel (SDS-PAGE) by electrophoresis and subsequently electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature, the PVDF membrane was incubated overnight at 4 °C with the appropriate primary antibody for TLR7 (1:100; Novusbio, USA), MyD88 (1:200; Boster, China), IRF7 (1:1000; Abcam, USA), beclin 1 (1:200; Boster, China), SQSTM1 (1:200; Boster, China), EV71/CA16-VP1 (1:1000; Millipore, USA) or GAPDH (as a loading control, 1:10000; Abmart, China). Then, the membrane was extensively washed three times with TBST prior to incubation for 1 h at room temperature with the corresponding secondary antibody (Abmart, USA) at a dilution of 1:12,000. Finally, the membrane was again extensively washed three times with TBST and then visualized using enhanced chemiluminescence reagents (Beyotime, China) and X-ray films (Kodak, Japan).

The cell or tissue samples were lysed by a RIPA lysate buffer (Beyotime, Shanghai, China) containing 1 × protease inhibitor cocktail (Beyotime, Shanghai, China). Protein concentration was quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). The samples (20 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a PVDF membrane (BioRad, Hercules, CA, United States). Non-specific binding was blocked by incubation of the membranes in 5% defatted milk for 1 h at RT. The membranes were incubated overnight with primary antibodies at 4°C and with secondary antibody for 2 h at RT. After incubation, the membranes were washed three times with TBST. The ECL Kit (Fdbio, Zhejiang, China) was applied to the membrane, and the membrane was imaged using a ChemiDoc XRS + (BioRad, Hercules, California, United States). The gray intensity of the target band was analyzed by Image Lab software. The antibodies used are presented in Supplementary Table 2.

The cells were washed thrice with cold PBS after transfection for 48 h. Cells were harvested and lysed with RIPA buffer (Beyotime Biotechnology) containing with 1× protease inhibitor cocktail (Beyotime Biotechnology). After incubating on the ice for 20 min and centrifuging at 12,000 rpm at 4 °C for 20 min, protein lysates were collected in a new centrifuge tube. After quantification using a bicinchoninic acid kit (Beyotime Biotechnology), equal amounts of samples were denatured in protein loading buffer (NCM Biotech) and heated at 100 °C for 10 min. Protein samples were separated by 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). The membranes were blocked in 5% skim milk diluted in 1× Tris-buffered saline with Tween 20 (TBST) for 1 h and incubated at 4 °C overnight with primary antibodies against Flag (1:1000, 8146S, Cell Signaling Technology) or GAPDH (1:10,000, 60004-1-Ig, Proteintech). After 1 h incubation with the corresponding secondary antibodies (1:10,000, 7076S, Cell Signaling Technology) for 1 h at room temperature on the secondary day, the membranes were visualized using Amersham ImageQuant 800 (Cytiva, United States) after detection with BeyoECL Plus (Beyotime).