Ni nta

Wild type (w.t.) and mutant variants of A3G^CTD (residues 191–384) and w.t. A3A were subcloned into pET28b (Novagen) and expressed in ArcticExpress cells (Agilent Technologies Genomics) with 0.5 mM IPTG induction overnight at 16 °C (supplemented with 50 μM zinc acetate). The His-tagged A3A and A3G^CTD were purified using HisPur Cobalt Resin (Thermo Fisher Scientific) in a buffer containing 500 mM NaCl, 50 mM Potassium Phosphate pH 7.5 and 5 mM β-mercaptoethanol and dialyzed into a final buffer containing 150 mM NaCl, 20 mM Tris-HCl pH 7.0 and 1 mM tris(2-carboxyethyl)phosphine (TCEP). A3G^CTD was further purified by size exclusion chromatography.
Full-length A3G was subcloned into pcDNA3.1 (Thermo Fisher Scientific Invitrogen) and expressed with a C-terminal Myc/His tag in human embryonic kidney (HEK) 293 T cells, harvested 48 hours post transfection and purified as previously described26 (link). Cells were broken by 3 cycles of freeze-thaw in lysis buffer (50 mM Tris, pH 8.0, 1 mM PMSF, 10% (v/v) glycerol and 0.8% (v/v) NP-40) and soluble fraction was adjusted to 0.8 M NaCl and treated with 50 μg/ml RNaseA (30 min at 37 °C) prior to purification using Ni-NTA (Thermo Fisher Scientific). Elution buffer contained 50 mM Tris, pH 8.0, 0.3 M NaCl, 10% (v/v) glycerol, 250 mM imidazole and subsequently dialyzed into a final buffer containing 50 mM Tris pH 8, 0.3 M NaCl, 1 mM DTT.

For in vitro Ni-NTA pull-down assays, 10 μg of His₆-tagged Rev-A IN in 100 μL pull-down buffer (25 mM Tris HCl pH 7.5, 150 mM NaCl, 25 μM ZnCl₂, 0.1% (v/v) Nonidet P40, 20 mM imidazole) was mixed with 10 μL settled volume of Ni-NTA beads (Thermo Scientific) previously washed with pull-down buffer. Following incubation at 4°C for 2 h with gentle agitation, 10 μg BSA and 10 μg of BRD4_462–720 or BRD4_{462–720/L630E} were added, and mixtures were incubated overnight at 4°C. The beads were washed five times with pull-down buffer and briefly centrifuged for 1 min at 1,300 g, and were then resuspended in 20 μL 2X sodium dodecyl sulfate (SDS) gel loading buffer and boiled for 10 min. The resulting supernatant was analyzed by denaturing gel electrophoresis on a 10% acrylamide gel. Proteins were detected by staining with Coomassie blue.

The RNMT1, DXO1, nDXO1, and cDXO1 coding sequences were PCR-amplified from cDNA of WT, and the DXO1^K412Q mutant clone was previously reported^{26 (link)}. These fragments were cloned into the pET28a expression vector to generate the expression constructs. The GST-DXO1 fusion was cloned into the pGEX-4T-1 vector. The plasmids were transformed into E. coli BL21 Rosetta cells to express recombinant protein fused with the 6X His tag or GST tag at their N-terminus. To induce expression of these proteins, IPTG was added to cells when OD₆₀₀ = 0.6, and the cells were incubated at 28 °C for 3–4 h. The recombinant proteins were purified by Ni-NTA or GST agarose beads according to the manufacturer’s instruction (Thermo Fisher). The purified recombinant proteins were stored in a buffer containing 20 mM Tris (pH 7.5), 100 mM NaCl and 5% (v/v) glycerol at −20 °C until further use.

The full-length gene encoding RSV-A NP (GenBank: KT992094.1) was amplified by PCR with two pairs of primers, including the forward primer 5′-GGA TCC GAT GGC TCT TAG CAA AGT C-3′ and the reverse primer 5′-CTC GAG CAT AGG TTG TTC CCT TCA A-3′. The RSV-NP DNA fragment was sub-cloned into pET21b (+) and RSV rNP antigen expression was induced by 0.5 mM of isopropyl β-d-1-thiogalactopyranoside. Total proteins were harvested and purified through Ni-NTA (Thermo Fisher Scientific).
The expression of antigen was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an anti-mouse 6× his-tag antibody (Thermo Fisher Scientific) diluted 1:10,000. The membrane was then washed three times and incubated with the secondary anti-mouse antibody (anti-mouse IgG conjugated with horseradish peroxidase; Abcam) diluted 1:40,000 in blocking buffer for 30 min at room temperature. After washing five times, the protein bands were visualized by Bio-Rad ChemiDoc XRS+ (Bio-Rad) (Hercules, CA, USA).