Ab180606

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.

The total proteins were extracted and separated by 10% or 15% SDS-PAGE and were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Massachusetts, USA) using a semi-dry Gel Transfer Device (Bio-Rad, Hercules, California, USA). The membranes were blocked using 5% non-fat milk and probed with primary antibodies and HRP-conjugated secondary antibodies. Antigen-antibody complexes were visualised using a chemiluminescent ECL detection system and analysed using a ChemDoc XRS+image analyser. GAPDH or β-actin was used as an internal control. The antibodies used included anti-β5i (1:5000, ab180606, Abcam), anti-RIG-I (1:2000,3743, Cell Signaling Technology), anti-Phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology), anti-Phospho-IRF3 (1:500, ab76493, Abcam), anti- IFNβ (1:500, ab275880, Abcam), anti- MxA (1:500, sc-166412, Santa Cruz), anti-MuRF1 (1:2000, ab172479, Abcam), anti-β-actin (1:5000, YM3028, Immunoway) and anti-GAPDH (1:10000, YM3029, Immunoway). The selective β5i inhibitor PR-957 was purchased from Selleck Chemicals (Houston, Texas, USA).

Frozen muscle sections were incubated with H₂O₂ (3%) for 15 min, followed by washing three times with phosphate buffer solution Tween-20 (PBST). The slides were blocked with goat serum at room temperature for 2 hours. Rabbit antibodies against human β5i (1:2000, ab180606, Abcam) and RIG-I (1:500, ab45428, Abcam) were used as the primary antibodies. A monoclonal rabbit IgG (1:2000, ab37415, Abcam) was used as isotype control for β5i (1:2000, ab37415, Abcam) and a polyclonal rabbit IgG (1:500, ab172730, Abcam) was used as isotype control for RIG-I. After washing with phosphate-buffered saline (PBS), the slides were incubated with HRP-conjugated secondary antibodies. Each incubation step was followed by three washes with PBST. Diaminobenzidine was used as the chromogen to visualise the proteins.