Mouse anti mhc 1

Whole cell lysates were prepared with m-per (Pierce, Rockland, IL). In some cases, nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear Extraction Reagent kit (Pierce) with protease inhibitors (Halt Protease Inhibitors Cocktail, Pierce). Protein concentrations were determined using the BCA assay (Pierce). Samples were subjected to LDS-PAGE (4 to 12 % NuPAGE Tris-Bis) (Invitrogen, Carlsbad, CA) and transferred to a nitrocellulose membrane with iBlot (Life Technologies Grand Island, NY). Membranes were blocked overnight with 5% w/v nonfat dry milk in 1X TBST or Odyssey blocking buffer (Licor, Lincoln, NE). Primary antibodies used were: mouse anti-β-actin, mouse anti-β-tubulin, mouse anti-CRBN, or mouse anti-Ikaros (IKZF-1) (Sigma-Aldrich); mouse anti-IRF-4, rabbit anti-p21, mouse anti-MHC-I, mouse anti-Aiolos (IKZF-3) (Santa Cruz Biotechnology, Dallas, TX); mouse anti-open reading frame 45 (ORF45) (Abcam, Cambridge, UK), rabbit monoclonal antibody to vIL-6 [51 (link)], mouse anti-K3 (gift from Dr. Jae Jung, USC); or mouse anti-K5 (gift from Dr. Ueda, Osaka University). Secondary antibodies were conjugated to alkaline phosphatase (Promega, Madison. WI) or conjugated to green or red fluorescent dyes for use with LI-COR system.

The antibodies were listed above for flow cytometry at in vitro coculture assay. For immunoblotting, the following antibodies were used: mouse anti–MHC-I (Santa Cruz Biotechnology, sc-55582), rabbit anti-pSTAT1 (Cell Signaling Technology, 7649), rabbit anti-STAT1 (CST, 14994), rabbit anti-IFNGR1 (Millipore, MABF753), mouse anti–β-actin (Sigma-Aldrich, A5441), rabbit anti–RIG-I (Cell Signaling Technology, 3743), rabbit anti–MDA-5 (Cell Signaling Technology, 5321), rabbit anti-MAVS (Cell Signaling Technology, 3993), rabbit anti-pIRF3 (Cell Signaling Technology, 29047), rabbit anti-IRF3 (Cell Signaling Technology, 4302), rabbit anti-p-p65 (Cell Signaling Technology, 3033), rabbit anti-p65 (Cell Signaling Technology, 8242), rabbit anti-pSTING (Cell Signaling Technology, 72971), and rabbit anti-STING (Cell Signaling Technology, 13647). rabbit anti-p65 (Cell Signaling Technology, 8242) was used for RIP and ChIP, and normal rabbit immunoglobulin G (IgG; Cell Signaling Technology, 2729) was served as a negative control.