Ab126630

The antibody used to detect dsRNA was a murine MAb pan-Enterovirus clone 9D5 purchased from Millipore Sigma (cat#3361, ready for use, further diluted by dilution 1:2 in our lab). The rabbit MAbs against PKR (1:200, ab32506), p-PKR (1:100, ab81303) and MDA-5 (1:250, ab126630) were purchased from Abcam. The secondary antibodies, Goat-anti-mouse Alexa Fluor 488 (1:2,000) and Donkey-anti-rabbit Alexa Fluor 594 (1:2,000) were purchased from Invitrogen. The RIG-I antibody conjugated Alexa-594 was purchased from Santa Cruz (1:1,000, sc-376845). A murine monoclonal anti-JUNV NP antibody was obtained from BEI (NA05-AG12) and conjugated to Alexa 647 dyes (1:1,000, Invitrogen). RNase III was purchased from Life technologies (AM2290) and RNase I from Promega (M4261).

Cells were grown on coverslips (13 mm), washed with 1 mL PBS, and fixed for 10 min in 4% paraformaldehyde in PBS (Affymetrix, High Wycombe, UK). Cells were washed three times with PBS-T for 5 min, followed by permeabilization with 0.25% Triton-X-100 (Sigma) in PBS for 15 min. Samples were washed three times in PBS-T and blocked with ready-made 10% normal goat serum (Life Technologies, Paisley, UK) for 1 h at room temperature. Primary antibodies were diluted in blocking solution: J2 (monoclonal mouse IgG2a kappa chain; Scicons, Susteren, The Netherlands) and TOM20 (Abcam, ab186734) at 1:200 dilution, PKR (monoclonal IgG rabbit anti-human, Abcam, ab32052), MDA5 (monoclonal IgG rabbit anti-human, Abcam, ab126630) at 1:1000. After incubation at RT for 1 h cells were washed (three times 5 min in PBS-T) followed by secondary antibody incubation: For J2, an Alexa fluor™ 488 coupled goat anti-mouse IgG2a (Thermo) was used, otherwise an Alexa fluor™ 594 goat anti-rabbit IgG H + L (Thermo). Both secondary antibodies were diluted in blocking buffer 1:1000 and incubated for 1 hr at RT in the dark. After three washes, the cells were stained with DAPI (Vector Laboratories, Newark, CA, USA) and mounted on slides with ProlongTM Glass Antifade hard mounting medium (Thermo). Cells were imaged using an LSM800 Airyscan confocal microscope (Zeiss, Jena, Germany).