Flag immunoprecipitation kit

Primary murine cardiac fibroblasts were transfected with Adv-GFP-H1.0-FLAG or Adv-GFP for 48 h. The co-IP assay was performed using a FLAG Immunoprecipitation Kit (Sigma-Aldrich, FLAGIPT1-1KT) according to the manufacturer’s instructions. Immunoprecipitated proteins were eluted using the SDS sample buffer included in the kit and then subjected to immunoblotting.

To map the interacting domains between 3D^pol and Prp8, the full-length and various truncated forms of human Prp8 were amplified by PCR from the human Prp8-pCMV-XL5 cDNA clone (OriGene) using specific primers. The PCR product was inserted into a pCMV-HA vector (Clontech) between the XhoI and NotI sites to enable the expression of HA-tagged proteins. The EV71 full-length infection cDNA clone was used to amplify full-length and various truncated forms of EV71 3D^pol by PCR, followed by cloning into the EcoRI and KpnI sites of the p3XFLAG-Myc-CMV-25 vector (Sigma) to enable expression of the EV71 3D^pol constructs as fusions with 3 adjacent FLAG epitopes. To overexpress these proteins, 2 µg of the constructs of the various truncated forms of Prp8 and 3D^pol was co-transfected into HEK293T cells (1×10⁶/per 6-well plate) using Lipofectamine 2000 reagent (Invitrogen) for 48 h. The cells were harvested for FLAG-IP using a FLAG-immunoprecipitation kit (Sigma). After lysis and centrifugation, the supernatant was treated with 10 µg/ml RNase A at 30°C for 1 h, and then 40 µl of anti-FLAG M2 agarose affinity gel was added at 4°C for 12 h. Proteins were then eluted by competition with 3×FLAG peptide. In the WB assay, the precipitated proteins were identified using an anti-HA antibody (diluted 1∶5000; Sigma) and an anti-FLAG antibody (diluted 1∶5000; Sigma).

cDNAs were synthesized from antennal or testis mRNA (a gift from F. Newton, University of Edinburgh, Edinburgh, Scotland, UK) and cloned into the C-terminal site of plasmids pAWH (3x HA epitopes) and pAWF (3x FLAG epitopes) of the Drosophila Gateway Vector collection (Carnegie Institution for Science). Primers for synthesis are in Table S4. Transfection into S2 cells was performed using X-TREME GENE HP DNA transfection reagent (Roche). The cells were harvested after 48–72 h, and coIP was performed according to the FLAG Immunoprecipitation kit (Sigma-Aldrich). After Western blotting, the polyvinylidene fluoride membrane was incubated with mouse anti-FLAG M2 antibodies (1:1,000; F1804; Sigma-Aldrich) and rabbit anti-HA (1:4,000; ab9110; Abcam) antibodies. Secondary antibodies were supplied by Li-COR (IR Dye 680RD and IR Dye 800CW), and protein detection was performed on a Li-COR Odyssey scanner using ImageStudio v5.2 software.

Polyclonal antibodies (Abs) against zebrafish TBK1 were generated according to our previous method (18 (link)), which can recognize TBK1 normal form, TBK1_tv1 and TBK1_tv3 isoforms, but not for TBK1_tv2 isoform. Anti-FLAG and anti-HA mouse Abs, dimethylsulfoxide (DMSO), and FLAG Immunoprecipitation Kit were purchased from Sigma-Aldrich. The anti-pTurboGFP antibody was purchased from Everogen. The anti-GAPDH mouse antibody was purchased from Proteintech. Poly(I:C) was purchased from InvivoGen. The MG132 and NH₄Cl were purchased from Selleck. Lipofectamine 2000 and protease inhibitor cocktail were purchased from Thermo Fisher Scientific.