Sc 376845

To explore the colocalisation of β5i and RIG-I, double-label immunostaining was performed. After blocking with 5% bovine serum albumin in PBST at room temperature for 1 hour, the samples were treated with mouse anti-RIG-I monoclonal antibody (1:50, sc-376845, Santa Cruz Biotechnology) and rabbit anti-β5i monoclonal antibody (1:2000, ab180606, Abcam) overnight at 4°C, followed by three washes with PBS (10 min each wash). Next, the samples were treated with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, 8890, Cell Signaling Technology) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, 4412, Cell Signaling Technology), which were used as the secondary antibodies, in a dark chamber for 1 hour, followed by three washes with PBST. After counterstaining with DAPI (Beyotime, Shanghai, China), the slides were mounted with coverslips. To clarify whether β5i is expressed in regenerative myocytes, we performed double-label immunostaining using rabbit anti-β5i monoclonal antibody (1:2000, ab180606, Abcam) and mouse anti-NCAM1 monoclonal antibody (1:500, ab6123, Abcam), following the same procedure mentioned above. In addition, double-label immunofluorescence staining using rabbit anti-β5i monoclonal antibody and mouse anti-MxA monoclonal antibody (1:100, sc-166412, Santa Cruz) was performed following the above procedure.

Mouse lung tissues and A549 ​cells were lysed in RIPA buffer as mentioned for Western blotting. The lysates were centrifuged at 12,000×g and 4 ​°C for 15 ​min. Supernatants were collected, and 400 ​μg lysate samples were incubated with 2 ​μg of an anti-MAVS primary antibody overnight at 4 ​°C in a rotator. Then, 20 ​μL Protein A/G agarose (Beyotime Biotechnology, Shanghai, China) was added and incubated for an additional 3 ​h at 4 ​°C, after which the lysates were washed 5 times with PBS and subjected to Western blot analysis with primary antibodies specific for MAVS (Abcam, Source: rabbit, Catalog: ab31334, Abcam, Cambridge, MA, USA) and RIG-I (Santa Cruz, Source: mouse, Catalog: sc-376845, Santa Cruz, CA, USA).