F3165

For immunofluorescence, antibodies against Tubulin (Abcam; ab6160), Flag (Sigma; F7425), Tubulin (Sigma; T4026), GFP (Roche; 1181446001), SKAP (Atlas; HPA042027), and CREST anti-sera (Europa; FZ90C-CS1058) were used. Images of immunostained cells were acquired using 100 times NA 1.4 objective on a DeltaVision Core microscope equipped with CoolSnap HQ Camera (Photometrics). For immunoblotting, antibodies against Tubulin (Sigma-Aldrich; T6557), EB1 (BD; 610534), flag (Sigma; F3165), EB3 (Millipore; AB6033), Cyclin-B (BD; 554176) were used. Immunoblots were developed using fluorescent secondary antibodies (LI-COR) and fluorescent immunoblots were quantified using the Odyssey (LI-COR) software. For allowing the merging of images from two different fluorescent channels of a single blot as in Fig. 1H, mouse anti-flag and rabbit anti-SKAP antibodies were used.

Immunoprecipitation was carried out with 500 μg cell lysate for endogenous PDE4D7 or DHX9, and overexpressed DHX9‐FLAG or PDE4D7‐VSV cell lysate incubated overnight in Protein G Sepharose beads (Invitrogen, Waltham, MA, USA) with 1 μg·μL⁻¹ of the appropriate antibody or IgG control; anti‐DHX9 (Abcam, #ab26271), anti‐PDE4D7 (Baillie Lab, University of Glasgow, Glasgow, UK), anti‐VSV (Abcam, Cambridge, UK, #ab1874), anti‐FLAG (Sigma‐Aldrich, #F3165), mouse IgG (Millipore, Burlington, MA, USA, NI03), rabbit IgG (Millipore NI01), sheep IgG (ThermoFisher #31243). Samples were centrifuged four times at 500 × g for 3 min prior to elution of bound protein complexes in 2 × Laemmli buffer for 5 min at 95 °C. Samples were analysed via SDS/PAGE and immunoblotting, then imaged via LI‐COR Odyssey (Lincoln, NE, USA).

Cells were lysed at 4°C for 10 min using NP-40 lysis buffer (50 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 1% (v/v) Igepal), supplemented with protease inhibitor cocktail (Merck). Cells were sonicated with Q700CA Sonicator (Q Sonica) on 50 amplitude for 5 s on/off for two cycles. Protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane. The membrane was blocked in Odyssey TBS Blocking Buffers (LI-COR Bioscience) for 1 hr at room temperature and subsequently incubated with specific antibody against FLAG (1:1000, Merck F3165) overnight at 4°C and HA (1:1000, Merck 05–902R) and GFP (1:1000 Santa Cruz Biotechnology sc-9996) 1 hr at room temperature. Afterwards, the membrane was washed and incubated with fluorescent Goat-anti-Rabbit and goat-anti-Mouse (1:2000; LI-COR Bioscience) for 1 hr at room temperature. Protein detection was performed in LI-COR Odyssey CLx (LI-COR Bioscience).

Anti-His₆; mouse monoclonal at 1:10 000 dilution (631212, Merck)
Anti-FLAG M2; mouse monoclonal at 1:10 000 dilution (F3165, Merck)
HA-Tag antibody; mouse monoclonal at 1:10 000 dilution (sc-7392, Santa Cruz Biotechnology)
Anti-Myc tag antibody; mouse monoclonal at 1:10 000 dilution (ab32, Abcam)
CaptureSelect Biotin Anti-C-tag conjugate; camelid antibody fragment at 1:10 000 dilution (7103252100, Thermo Fisher Scientific)
C.tag HRP-conjugate; nanobody at 1:20 000 (in house)

BL21 (DE3) E. coli cells were co-transformed with both pET17-twin-strep-C16 and pET28b-flag-C4 and grown in standard LB medium supplemented with ampicillin and kanamycin. Protein expression was induced upon the addition of IPTG at 0.5 mM final concentration once cells had reached an optical density (600 nm) of 0.8–1.0 and the temperature had been reduced from 37 to 16 °C. After growth overnight, cells were pelleted by centrifugation and then resuspended in buffer A (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP), supplemented with lysozyme, benzonase nuclease (Merck Life Sciences) and cOmplete^TM EDTA-free protease inhibitor (Roche). Cells were lysed by sonication and cleared by centrifugation.
Pull-downs were performed using the cell lysate from the C16 and C4 co-expression. The lysate was divided in half, and then applied to either Strep-Tactin beads (IBA, catalog number 2-1613-002, 1 ml to obtain a bed volume of 50 µl) or Anti-Flag beads (Merck, catalog number M8823, 100 µl to obtain a bed volume of 50 µl). After extensive washing, proteins retained on the beads were eluted with the addition of either biotin or flag peptide respectively and the eluted proteins analysed by SDS-PAGE and immunoblotting with either anti-strep (Merck, catalog number SAB27002216, 1:7000 dilution) or anti-flag antibodies (Merck, catalog number F3165, 1:5000 dilution).

Transfection with plasmids that expressed RABV proteins was performed using Metafectane reagent, following the manufacturers’ instructions (Biontex). We added 500 nM 17-AAG to the transfected cells 6 h after infection, and the incubation was terminated 48 h after transfection. The protein extracts prepared with the RIPA buffer were resolved on 10% PAGE-SDS gels to detect myc-tagged G, M, N, and P proteins. 3xFLAG-L protein was resolved on 8% PAGE-SDS gel. The insoluble protein was separated by centrifugation for 20 min at 14,000× g and the pellet was solubilized in 8M urea before electrophoresis. The proteins were detected using monoclonal antibodies specific for Flag (Sigma, F3165) and myc (Merck, MABE282). A goat anti-mouse IgG-HRP antibody was obtained from Bio-Rad (cat. no. 170-6516). The original pictures were taken with a CCD camera with the exposure time adjusted to avoid pixel saturation. The registered signals were within the linear response range of the camera.