High capacity streptavidin agarose resin

RPMI, DMEM, and FBS were from Invitrogen. Hygromycin B and puromycin were from Gibco. Primary antibodies used were anti-HA (Cell Signaling Technology, catalog number C29F4), anti-pHistoneH3 (S10) (Abcam, catalog number ab14955), anti-LaminA/C (BD Biosciences, catalog number 612162), anti-GAPDH (Sigma-Aldrich, catalog number CB1001), and anti-β-actin (Santa Cruz Biotechnology, Inc., catalog number sc-1616). Secondary HRP-conjugated antibodies used were goat anti-rabbit (Cell Signaling Technology, catalog number 7074S) horse anti-mouse (Cell Signaling Technology, catalog number 7076S) and donkey anti-goat (Santa Cruz Biotechnology, Inc., catalog number sc-2020). Chemiluminescence reagent for Western blotting was SuperSignal™ West Pico PLUS. Mounting media for fluorescence microscopy was ProLong® Gold Antifade Reagent with DAPI. LPA supplied in chloroform was from Avanti Polar Lipids {Acyl-linked 18:1 lysophosphatidic acid [1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt)]}. For sulfenylation studies, DCP-Bio1 was from by Xoder Technologies (Winston Salem, NC), sepharose CL-4B resin was from Sigma, and high capacity streptavidin agarose resin was from Thermo Scientific. Propidium iodide and the nucleotides GTP and dATP were from Invitrogen.

HEK293T cells (RRID: CVCL_0063), within 25 passages, were seeded (9 × 10⁶ cells/dish) on 145 mm cell culture dishes (639160, Greiner Bio-One), cultured in DMEM (08458-16, Nacalai Tesque) containing 10% FBS and 0.5% penicillin–streptomycin solution. Following 48 h culture, plasmid encoding Fc-SBP and LOTUS-Fc-SBP were transfected with lipofection reagent (Polyethylenimine Max, 24765, Polysciences) and cultured for an additional 4 days. The culture medium was ultracentrifuged at 117,000×g for 1 h. Subsequently, the supernatant was added to streptavidin beads (High Capacity Streptavidin Agarose Resin, 20361, Thermo Fisher Scientific). SBP-fused protein was eluted from the beads with PBS containing 2 mM biotin. These proteins were stored at − 80 °C until use. The protein sample was prepared with 4× Laemmli buffer (40% glycerol, 8% SDS, 250 mM Tris–Cl pH 6.8, and 0.06% bromophenol blue) containing 10% β-mercaptoethanol and boiled for 7 min to determine the concentration of purified proteins. Each sample was electrophoresed on a Tris–glycine SDS polyacrylamide gel, and the gel was incubated with Coomassie brilliant blue R-250 (031-17922, Wako Pure Chemical Industries). The intensity of the stained protein was measured for each concentration using an ImageQuant LAS 4000 mini instrument with ImageQuant TL software.

Wild-type and rlmH::ISS1 strains containing the pull-down plasmid pLNRK-SA were subcultured 1:20 into 100 mL GM17 Cam₁₀, grown to an OD₆₀₀ of 0.5 and induced for 3 h with nisin. Following induction, 50 mL aliquots of cell culture were collected by centrifugation and pellets were stored at − 80 °C. Cell pellets were resuspended in 300 μL CB₅₀₀ buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1 mM EDTA) and lysed by sonication. High Capacity Streptavidin Agarose Resin (Thermo Scientific) was washed 3 times with CB₅₀₀ and then incubated with lysate overnight, nutating at 4 °C. The resin was then washed 10 times with CB₅₀₀, followed by elution with 500 μL of 10 mM biotin in CB₅₀₀. RNA was isolated from eluate with two rounds of PCIA extraction followed by ethanol precipitation. Northern blots were performed on isolated RNA as described above (1 h hybridizations), with identical gels run side-by-side to perform a single blot for each probe (IDT1073 for intron RNA and IDT861 for 16S rRNA). Statistical significance was assessed with a t-test.

High Capacity Streptavidin Agarose Resin (Thermofisher) was incubated at 37 °C for 1 hour while tumbling (Labnet, revolver) with 1% (w/v) BSA in 1× PBS (prepared from phosphate buffered saline tablets, Sigma Aldrich) in 1 : 4 volume ratios. 20 μL beads were used per reaction to immobilize RPA product from one RPA reaction tube (Twist Dx basic kit).

HEK293T cells were grown in 10-cm dishes and co-transfected with expression plasmids for IRF9-S2C, and V5-NiV-V, V5-C6 or V5-GFP using calcium phosphate transfection in triplicate for each condition. Sixteen hours later cells were lysed in lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 5% glycerol and protease (cOmplete Mini, Roche) and phosphatase (PhosSTOP, Roche) inhibitors). Lysates were incubated firstly with 10 ng/ml poly(dI:dC) for 30 min, then with 100 pmol biotin-labelled ISREcore or biotin-labelled control DNA for 1.5 h and finally with 30 μl High Capacity Streptavidin Agarose Resin (Thermo Scientific) for 3.5 h. Immunoprecipitations were washed four times with lysis buffer and proteins were eluted by boiling in buffer containing SDS. Samples were then analysed by SDS-PAGE and immunoblotting with the stated antibodies.