Western lightning chemiluminescence kit

Western blotting was performed as previously described[60 (link)]. Briefly, cell lysates were prepared from transfected cells using radioimmunoprecipitation assay (RIPA) lysis buffer, heated at 100°C for 10 min and were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, protein samples were electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and blocked for 2 h in Tris-buffered saline (10 mM Tris-HCl, pH 8.0, containing 150 mM NaCl) containing 5% (w/v) non-fat dry milk and 0.5‰ (v/v) Tween-20. The membranes were incubated with the primary antibodies overnight at 4°C, followed by incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The presence of HRP was detected by using a Western Lightning chemiluminescence kit (Amersham, USA) according to the manufacturer’s protocol.

Total cell protein lysates were extracted from the transfected 293T cells with CA630 lysis buffer (150 mM NaCl, 1% CA630 detergent, 50 mM Tris base [pH 8.0]). Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA). Each PVDF membrane was blocked with 0.1% Tween 20 and 5% non-fat dry milk in Tris-buffered saline and subsequently incubated with a primary antibody. The primary antibodies were specific for influenza A virus PA (1:3,000, GeneTex, USA), influenza A virus PB1 (diluted 1:3,000, GeneTex, USA), influenza A virus NP (1:3,000, GeneTex, USA), and influenza A virus PA-X (diluted 1:2,000, polyclonal rabbit antiserum against the PA-X derived peptide (CAGLPTKVSHRTSPA) (diluted 1:3,000 Genscript, China). The secondary antibody used was either horseradish peroxidase (HRP)-conjugated anti-mouse antibody or HRP-conjugated anti-rabbit antibody (diluted 1:10,000 Beyotime USA) as appropriate. HRP presence was detected using a Western Lightning chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany), following the manufacturer's protocol.

Total cell protein lysates were extracted from transfected 293T cells or infected MDCK cells with CA630 lysis buffer (150 mM NaCl, 1% CA630 detergent, 50 mM Tris base [pH 8.0]). Cellular proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Germany). Each PVDF membrane was blocked with 0.1% Tween 20 and 5% nonfat dry milk in Tris-buffered saline and subsequently incubated with a primary antibody. Primary antibodies were specific for influenza A virus PA (1:3000, GeneTex, USA), influenza A virus PB1 (diluted 1:3000, Thermo Fisher Scientific, USA), influenza A virus PA-X (diluted 1:2000, was kindly provided by Dr. Xiufan Liu, YangZhou University, China). The secondary antibody used was either horseradish peroxidase (HRP)-conjugated anti-mouse antibody or HRP-conjugated anti-rabbit antibody (diluted 1:10,000 Jackson ImmunoResearch USA), as appropriate. HRP presence was detected using a Western Lightning chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany), following the manufacturer's protocol.