Anti ha polyclonal antibody

The protein samples were boiled with 1 × SDS or 5 × SDS sample loading buffer for 10 min and analyzed with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After being transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA), the membranes were blocked in 5% non-fat milk in 1 × Tris-buffered saline with 0.1% Tween 20 (TBST) and then incubated with diluted anti-His monoclonal antibody (SAB, Fairfield, NJ, USA), anti-VP28 monoclonal antibody, anti-β-actin monoclonal antibody (TransGen Biotech, Beijing, China), anti-FLAG monoclonal antibody (ABclonal Technology, Wuhan, China) or anti-HA polyclonal antibody (Proteintech Group, Chicago, IL, USA) at room temperature for 1 h, respectively. After washing with TBST three times, goat anti-mouse IgG monoclonal antibody conjugated to horseradish peroxidase or goat anti-rabbit IgG polyclonal antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies. After a final wash with TBST three times, the membrane was exposed using an Enhanced Chemiluminescent Kit (NCM Biotech, Suzhou, China).

Samples were separated by SDS‐PAGE with 10% polyacrylamide gel and stained with Bio‐Safe Coomassie stain (Bio‐Rad, Hercules, CA, USA). For Western blot analysis, samples were separated by 8% polyacrylamide gel and transferred to Hybond‐ECL membrane (GE Healthcare, Chicago, IL, USA). The membrane was treated with anti‐HA polyclonal antibody (Proteintech, Rosemont, IL, USA) and anti‐rabbit IgG HRP‐linked antibody (GE Healthcare), or PGK1 monoclonal antibody (anti‐PGK 22C4D8; Thermo Fisher, Waltham, MA, USA) and anti‐mouse IgG HRP‐linked antibody (GE Healthcare). Signals were developed with ImmunoStar LD (Wako, Osako, Japan) and detected by LAS‐3000 Mini digital imaging system (Fujifilm, Tokyo, Japan).