Ibright 1500 image system

The preparation of the cell lysates, SDS-PAGE and Western blot analysis were performed according to standard protocols. The PVDF membrane was incubated respectively with primary antibody (Table 1) overnight at 4°C on shaker. After incubating with HRP-linked secondary antibody (1:2000, Invitrogen, 31,430 and 32,460) for 1 hour at room temperature, the protein bands were detected using the iBright 1500 Image system (Thermo Fisher Scientific, Pittsburgh PA, United States) and assessed the ratio of target protein against house-keeping protein.

SDS-PAGE and western blot analysis were performed as previously reported (28 (link)). The following primary antibodies diluted in Can Get Signal^® Immunoreaction Enhancer Solution 1 (Toyobo Life Science) were used: Anti-RIG-I (1:3,000), anti-MDA5 (1:3,000) and anti-β-actin (1:4,000). Following overnight dilution at 4˚C, the membrane was reacted with secondary antibody (1:10,000) diluted in Can Get Signal^® Immunoreaction Enhancer Solution 2 (Toyobo Life Science) for 1 h at room temperature and detected using Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.). Images were captured by cool saver AE-6955 (ATTO Corporation) or iBright 1500 Image system (Thermo Fisher Scientific, Inc.).