Rabbit anti rig 1 d14g6

Neutrophils (3 × 10⁶) from blood were isolated by percoll density gradients as previously described [3 (link)] and were lysed with a boiling Laemmli buffer. Samples were loaded onto a 15% SDS-PAGE gel. Proteins were then transferred onto nitrocellulose membranes using Bio-Rad’s Trans-Blot Turbo, which were blocked using 5% nonfat dry milk in 1X TBS-T buffer for 1 h at room temperature. The membranes were incubated overnight at 4 °C under mild agitation in 5% nonfat dry milk in 1X TBS-T buffer containing the following primary antibodies: mouse anti-Caspase-1 (p20) (mAb (Bally-1); Adipogen; AG-20B-0048-C100; 1:1000), mouse anti-Caspase-4 (MBL cat. M029-3; 1:1000), rabbit anti-GSDMD (Abcam, cat. ab215203; 1:1000), rabbit anti-α-actin (Sigma-Aldrich, cat. A2066; 1:5000), mouse anti-α-actin (Cell Signaling, cat. 3700; 1:1000), rabbit anti-RIG-I (D14G6) (Cell Signaling, cat. 3743S; 1:1000). Membranes were washed in 1x TBS-T and incubated with appropriate secondary HRP-conjugated antibodies diluted in 5% nonfat dry milk in 1X TBS-T buffer. Protein detection was done using an ECL™ Prime Western Blotting System (GE Healthcare) and an Amersham Imager 600 (GE Healthcare).

The pCMV6-Entry-Myc-DDK-RNF122 (RC210868), pCMV6-Entry-Myc-DDK-MEX3A (RC215359), pCMV6-Entry-Myc-DDK-MEX3C (RC221125), pCMV6-AC-GFP-DDX58 (RG217615), non-effective Scrambled shRNA Cassette in pGFP-C-shLenti Vector (shCTR, TR30021), pGFP-C-shLenti-MEX3A (TL308061B (#1) and TL308061C (#2)) were purchased from Origene (Rockville, MD, USA).
Mouse anti-RIG-I D-12 (sc-376845, 1:1000 for WB, 1:100 for IHC), anti-RIG-I HRP conjugated D-12 (sc-376845, 1:2000), mouse anti-HA-probe F-7 HRP (sc-7392 HRP, 1:1000) and mouse anti-β-Actin C4 (sc-47778, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Flag M2 HRP (A8592, 1:1000) was purchased from Sigma-Aldrich. Rabbit anti-RIG-I (D14G6, 1:1000) and Rabbit anti-PARP (95426S) were purchased from Cell Signaling (Beverly, MA, USA). Rabbit anti-MEX3A (ab79046, 1:1000 for WB, 1:100 for IHC) was purchased from Abcam (Cambridge, UK). HRP-conjugated secondary antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA).
Where indicated, cells were treated with MG132 (50 µM; Calbiochem, Nottingham, UK) for 4 h, Cycloheximide (100 µM, Sigma Aldrich) at indicated time.

Rabbit polyclonal antibodies against NS1 were obtained by immunizing animals with purified hexahistidine-tagged NS1 (His-NS1) (Zheng et al., 2017 (link)). Mouse monoclonal antibodies against M1 and rabbit polyclonal antibodies against NP were obtained as previously described (Koestler et al., 1984 (link)). Mouse anti-c-Myc (9E10) and mouse anti-FLAG (M2) antibodies were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-c-MYC antibodies and FLAG beads were purchased from Sigma. Rabbit anti-RIG-I (D14G6) was purchased from Cell Signaling Technology. Anti-GAPDH and anti-β-actin antibodies were purchased from Boao Rui Jing Technology Development Co., Ltd (Beijing). All secondary antibodies were obtained from Bai Hui Zhong Yuan Biotechnology. The lipofectamine reagent was purchased from Invitrogen. Opti-MEM was purchased from Gibco. The protease inhibitor cocktail was purchased from Roche. The Tet-On 3G Inducible Expression System was purchased from Clontech.