Anti ns1

The PB2 or NS1 plasmids were co-transfected with synthetic MIR2911 or ncRNA into HEK293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Samples of cultured cells were lysed in RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5); the lysates were resolved by 10% SDS-PAGE, transferred to a PVDF membrane (Millipore, Bedford, MA, USA) and probed with anti-PB2, anti-NS1 or anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruze, CA, USA).

Protein lysates (30 µg total protein) were prepared using RIPA buffer (Sigma-Aldrich) and then separated by SDS-PAGE on 10~15% acrylamide gels and transferred to polyvinyldifluoride (PVDF) membranes. The membranes were then incubated in a blocking buffer comprised of 5% (w/v) BSA, 0.2 M Tris base, 1.36 M NaCl, and 0.1% Tween 20 (TBS/T) for 1 h at room temperature and washed three times with 5 mL of TBS/T for 5 min each wash. Membranes were incubated overnight with the primary antibodies against p21, STING, phospho-TBK, total TBK, p-IRF3, IRF3, β-actin (Cell Signaling Technology) at 4℃. For influenza A virus protein expression, anti-NS1 (Santa Cruz Biotechnology), and anti-NP antibodies (Sino Biological Inc., Beijing, China) were used. VZV gE antibody (Abcam, Cambridge, MA, USA) were used to measure the expression of VZV protein. After washing three times with TBS/T, the membranes were incubated with HRP-conjugated anti-rabbit or mouse IgG secondary antibody (Cell Signaling Technology) for 1 h at 25℃. After washing three times with TBS/T, membranes were incubated with Western Lumi Pico solution (ECL solution kit) (DoGen, Seoul, Korea), and exposed to film.