Hrp conjugated anti mouse antibody

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.

Proteins were extracted from 7-day-old seedlings using an extraction buffer containing 50 mM Tris–HCl (pH 7.5), 10 mM EDTA, 2 mM EGTA, 0.1% SDS, 1 mM DTT mixture, and 0.1 mM PMSF. After centrifugation at 12,000 rpm and 4°C for 10 min, the supernatant containing the total protein was collected. For Western blotting, we first denatured proteins at 96°C in 5× SDS-PAGE loading buffer (1:4). These proteins were run on a 12% SDS-PAGE gel, and then was transferred to a nitrocellulose membrane via blotting. The blot was probed with mouse anti-GFP monoclonal antibody (1:1200), and the GFP signal was detected using an HRP-conjugated anti-mouse antibody (1:1000) (Beyotime Biotechnology). Intracellular TUB3 was used as a loading control (Qi et al., 2020 (link)). Protein molecular weight markers (20350ES72; YEASEN, China) were used as size standards in gel electrophoresis. The membranes were visualized using an enhanced chemiluminescence substrate and a Tanon 5200 multi-imaging system (Shanghai, China).