Glutathione sepharose

Spire-KIND (amino acids 1–234) was expressed as a GST fusion protein in bacteria, and purified on glutathione-sepharose (GE Biosciences, Buckinghamshire, UK) followed by Superdex200 gel filtration (GE Biosciences) of the glutathione-eluted GST-fusion protein. GST-KIND or GST alone was re-bound to glutathione-sepharose in binding buffer (50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM Hepes-HCl pH 7.4, 1 mM DTT, 0.02% thesit (Sigma, St. Louis, MO), 10 μg/ml aprotinin, 2 μg/ml leupeptin, 0.5 mM benzamidine). INF2-CT (amino acids 469–1249, containing FH1, FH2 and C-terminal regions) and INF2-NT (amino acids 1–420, containing DID and dimerization region) were purified as described (Ramabhadran et al., 2012 (link)). Proteins were mixed at 20 μM GST protein, 1 μM INF2-CT and 10 μM INF2-NT in binding buffer and incubated overnight, then quickly washed once in binding buffer. Proteins in glutathione sepharose-bound pellet were resolved by SDS-PAGE.

The following reagents were used: β-NAD⁺ (Sigma), ³²P-NAD⁺ (Perkin Elmer), interferon α (Peprotech), lipopolysaccharid (LPS) of E. coli (Sigma), 12-O-Tetradecanoylphorbol-13-acetate (PMA) (Sigma), protease inhibitor cocktail (PIC) (Sigma), H₂O₂ (Merck KGaA), Glutathione-sepharose (Sigma), TALON metal affinity resin (BD Bioscience), ADPr (Adenosine 5′ diphosphoribose sodium-salt, Sigma), anti-HA (3F10, Roche), anti-HA (Covance), anti-PAR (Trevigen), anti-α-Tubulin (Sigma), anti-ARTD10 (5H11)2 (link), anti-ARTD10 purified rabbit antibodies10 (link), anti-Actin (C4, MP Biomedicals), anti-FLAG (Sigma), anti-GFP (Rockland), anti-MCM2 (N-19, Santa Cruz), goat anti-rat IgG (H + L) secondary antibody Alexa Fluor 555 conjugate (Invitrogen), anti-rabbit-HRP (Jackson Immunoresearch), anti-mouse-HRP (Jackson Immunoresearch), anti-rat-HRP (Jackson Immunoresearch). Rabbit polyclonal, purified ARTD8-specific antibodies were generated against the peptide NLVSDKIPKAKDTQG (aa 1193–1207).

The Escherichia coli (E.coli) strain BL21 was used to produce and purify the GST-tagged or His-tagged proteins. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to increase the protein expression final dosage of 1 mM into BL21 bacterial medium when the culture’s OD600 reached 0.5–0.7, then inducted at 16 °C in a 300 rpm shaker overnight. Bacteria cells were pelleted and resuspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 2 mM EDTA). Each sample was sonicated for 40 min using an EpiShearTM Probe Sonicator (pulse 6s on, 10s off, 40% amplitude) to disturb the integrity of the cells. After centrifuging the sample at 10,000 rpm for 40 min at 4 °C, the protein concentration was extracted from the supernatant. For the purification of GST-tagged or His-tagged proteins, BeyoGoldTM His-tag Purification Resin (Beyotime, Nantong, China) or Glutathione Sepharose (Sigma, St. Louis, MO, USA) were employed, respectively. Finally, loading buffer was added to each sample (1 mg), and the Bradford assay was performed. The appropriate proteins were treated with Glutathione Sepharose at 4 °C for 4 h to perform the GST pull-down. The samples were then Western blot tested after being washed three times with PBS and 1% TritonX-100 for a total of 1 h.

5′-pppRNA and poly(I:C) were purchased from InvivoGen, and were used at a final concentration of 0.5 µg/mL and 10 µg/mL, respectively. MG132, IFN-β, IFN-β ELISA kit, Glutathione-Sepharose, and the Dual-Luciferase Reporter Assay System were purchased from Sigma Aldrich, R&D Systems, PBL Interferon Source, GE Healthcare, and Promega, respectively. Antibodies against the following IAV proteins were generated in our Laboratory: PB2, PB1, PA, and NP. Anti-TRAF3 (sc-6933), anti-TRIM35 (sc-100880), and anti-ubiquitin (sc-8017) were from Santa Cruz. Anti-TRAF3 (4729), anti-MAVS (24930), anti-K63-linkage specific polyubiquitin (5621), anti-K48-linkage specific polyubiquitin (8081) were from Cell Signaling. Anti-Flag (F1804; F7425), anti-Myc (C3965; M4439), anti-V5 (V8012), and anti-TRIM35 (SAB2103161) were from Sigma Aldrich. Anti-IFITM3 (11714-1-AP), anti-HA (51064-2-AP), and anti-GAPDH (10494-1-AP) were from Proteintech. Anti-V5 (AB3792) was from Millipore. Anti-GFP (ab6556), and anti-GST (ab58626) were from Abcam. Mouse IgG (A7028) was from Beyotime. Alexa Fluor 633 goat anti-mouse IgG (H + L) (A21050) and Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (A21206) from Life Technologies were used for confocal microscopy. DyLight 800 goat anti-mouse IgG (H + L) (072-07-18-06) and DyLight 800 goat anti-rabbit IgG (H + L) (072-07-15-06) were from KPL.

NHE9 c-terminal (NHE9^c) was expressed as a complex of a glutathione S-transferase (GST)-fused human SLC9A9. Recombinant proteins fused with GST binding protein were expressed in BL21 Star (DE3) cells. The protein complex was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and purified with glutathione-Sepharose (Sigma). Bacterial from 500 ml of LB-medium (MP biomedicals, LLC) culture were sonicated and centrifuged, and the supernatant was applied to an amylose resin column equilibrated with phosphate-buffered saline (PBS) and incubated for 30 min at room temperature. The resin was washed 3 times with PBS, and cell lysate of Eca109 (10 dishes, 10 cm) was added to the resin. After incubation for 1 h at 4°C, the resin was washed 6 times with PBS, and bound proteins were eluted with sample buffer and analyzed by SDS-PAGE. The positive results were analyzed with surface- enhanced laserdesorption/ionization – time of flight – mass spectrometry (SELDI- TOF- MS) to detect the proteins. The same sample also prepared for western blotting identification using the corresponding antibodies.