Rabbit anti mx1

Proteins were separated in Mini-Protean TGX stain-free precast gels (Bio-Rad) at 120 V for 1 h and subsequently subjected to UV activation and quantitative gel imaging to compare the amounts of loaded protein. Proteins were transferred to nitrocellulose membranes using Trans-Blot Turbo transfer system (Bio-Rad). The primary antibodies used were rabbit anti-MX1 (1:1,000; Proteintech), rabbit anti-MX2 (1:1,000; Novusbio), rabbit anti-CypA SA296 (1:3,000; Biomol), mouse anti-alpha-tubulin (1:3,000; Sigma-Aldrich), rabbit anti-CPSF6 (1:3,000; Abcam), and rabbit anti-MAPK (Erk1/2; 1:1,000; Cell Signaling). Goat anti-mouse or anti-rabbit IgG secondary antibodies were coupled to horseradish peroxidase (Cell Signaling Technology), and proteins were detected using Pierce ECL Plus Western blotting substrate (Thermo Scientific).

Membranes were stained as described above with additional AnnexinV-BV421 staining (Milteny Biotec). Subsequently, cells were fixed and permeabilized by a permeabilization buffer set (eBioscience) with 1% paraformaldehyde, 0.5% saponin and stained with either rabbit anti-Mx1 (ProteinTech, Chicago, USA), rabbit anti-MDA5 (Abcam, Cambridge, UK), rabbit anti-DDX58 (Abcam), rabbit anti-IFI16 (Abcam) and rabbit anti-ZBP1 (Thermofischer, Rockford, USA)) and incubated in the dark for 45 min on ice. As a secondary antibody, chicken anti-rabbit-AF488 (Invitrogen, Carlsbad, USA) was used. Unstained cells and isotype-matched controls (Becton Dickinson Biosciences) were used to assess antibody specificity. Analysis was performed as previously described [21 (link)].