Anti flag and anti tubulin monoclonal antibody

Total EPC cellular or macrophages lysates were generated by using 1 × SDS-PAGE loading buffer, respectively. Proteins were extracted from cells and were measured with the BCA Protein Assay Kit (Vazyme) and then subjected to SDS-PAGE (10%) gel and transferred to polyvinylidence fluoride (Millipore, USA) membranes by semidry manner (Bio-Rad Trans Blot Turbo System) (35 (link)). The membranes were blocked for 1 h with 5% BSA. Then the membranes were incubated at 4°C overnight with anti-flag mouse mAb. Protein was blotted with different antibodies (Abs). The antibody against TRAF6 was diluted at 1: 400 (ProteinTech), anti-Flag and anti-Tubulin monoclonal antibody were diluted at 1:2,000 (Sigma), and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) was diluted at 1:5,000. The results were representative of three independent experiments. The immunoreactive proteins were detected by using WesternBright^TM ECL (Advansta). The digital imaging was performed with a cold charge-coupled-device camera.

Cellular lysates were generated by using 1 × SDS-PAGE loading buffer. Proteins were extracted from cells and measured with the BCA Protein Assay kit (Vazyme), then subjected to SDS-PAGE (10%) gel and transferred to PVDF (Millipore) membranes by semidry blotting (Bio-Rad Trans Blot Turbo System). The membranes were blocked with 5% BSA. Protein was blotted with different antibodies. The antibody against MAVS was diluted at 1: 500 (Abcam); anti-Flag and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); the anti-GFP monoclonal antibody was diluted at 1: 1,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000. The results were the representative of three independent experiments. The immunoreactive proteins were detected by using WesternBright^TM ECL (Advansta). The digital imaging was performed with a cold CCD camera.