Mammalian protein extraction reagent

Proteins were isolated using Mammalian Protein Extraction Reagent (CWBIO, Beijing, China) supplemented with protease inhibitors. The protein concentration was determined by a BCA Protein Assay Kit (CWBIO, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% nonfat dry milk for 1 h and then incubated with primary antibodies at 4°C overnight. After washes, relevant secondary antibodies were used at room temperature for 1 h. Afterwards, the membranes were washed and developed using standard chemiluminescence and the Bio-Rad ChemiDoc™ XRS+System.

H9c2 cells were lysed using Mammalian Protein Extraction Reagent (CWBIO, Beijing, China) and removed the cells debris through centrifuging for 15 min (15,000 rpm, 4 °C). Total protein was harvested and quantified using BCA assay kit (DINGGUO, Beijing, China). For each sample, approximately 20 μg proteins was separated by SDS-PAGE and transferred to a PVDF membrane. Then PVDF membrane was blocked in 5% non-fat dry milk for 2 h, followed by incubation with primary antibodies against Caspase-3 (1:1000 dilution, #14220, Cell Signaling Technology), Bcl-2 (1:1000 dilution, #3498, Cell Signaling Technology), Bax (1:5000 dilution, ab32503, Abcam), FAIM3 (1:1000 dilution, A6320, Abclonal, China), β-actin (1:1000 dilution; #4970, Cell Signaling Technology) at 4 °C overnight. Subsequently, the membrane was incubated with a secondary antibody (1:5000) at RT for 1 h. After washes, the immunoreactivity was detected using the standard chemiluminescence (Thermo-Fisher). The bands were quantified by measuring the band intensity for each group.

Total protein was abstracted from NPTr cells which were collected and lysed with Mammalian Protein Extraction Reagent (Cwbio, Beijing, China) after transfection and/or infection. Then, the concentration of total protein was measured using Bradford Protein Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Equal protein quantities were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (GE Life Science, Piscataway, NJ, USA). The membranes were blocked in 5% bovine serum albumin (BSA) for 1 h and then incubated with specific primary antibodies for 2 h at room temperature. After being washed three times, the membranes were incubated with relative second antibodies for 1 h. Finally, the protein blots were visualized using enhanced chemiluminescence reagent (Advansta, Menlo Park, CA, USA). The primary antibodies against β-actin and Horseradish peroxidase-conjugated anti-mouse/rabbit secondary antibodies were purchased from Abclonal Technology (Wuhan, China). Rabbit polyclonal antibody against viral NP was purchased from GeneTex, Inc. (San Antonio, TX, USA).