Primary v5 antibody

Cells were first seeded on to polylysine coated coverslips at a density of 10000/well and cultured overnight following which they were fixed in 4% paraformaldehyde for 30 min. The fixed cells were washed with PBS and then permeabilized for 10 min using 0.2% Triton X-100-PBS. The permeabilized cells were washed with PBS and then blocked for 30 min using the block solution (1% BSA–PBS). The cells were then incubated with the V5 primary antibody (mouse, Invitrogen, diluted 1/1000 in block solution) for 1 h at room temperature, washed with PBS and then incubated for 1 h with the AlexaFluor 488 secondary antibody (mouse, Invitrogen, diluted 1/2000 in block solution) and the nuclear stain Hoechst 33258 (Invitrogen, diluted 1/10000 in block solution). Finally, the cells were washed with PBS and the coverslips were mounted on to microscope slides. The Carl Zeiss LSM 710 microscope and the Carl Zeiss Zen software (version 1.0.1.0) were used to capture images of the immunostained cells.

To process the hare HN-R cells for immunofluorescence microscopy, cells were washed twice with PBS and fixed with 2% formaldehyde, prepared fresh in PBS from paraformaldehyde, for 12 min at room temperature (RT). The cells were then permeabilized for 1 min at RT in PBS containing 0.1% Triton X-100, washed twice with PBS, and blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at 37°C. The cells were then incubated with V5 primary antibody (Invitrogen, USA) at 4°C overnight, washed six times with PBS, and incubated with Alexa Fluor 633 anti-goat secondary antibody (Thermo Fisher, USA) for 30 min at 37°C. Nuclei and viral factories were then stained with 4′,6-diamidino-2-phenylindole (DAPI), and the coverslips were sealed with nail polish. Samples were imaged with a Nikon C2 scanning confocal or Nikon Ti microscope, using a 60× Plan Apo water immersion lens with a numerical aperture of 1.2. Excitation lasers of 405 nm, 488 nm, and 640 nm paired with blue, green, and far-red detectors were used to detect DAPI, GFP, and immunolabeled V5-tagged M159, respectively. Images were postprocessed using Nikon Elements software.

Transfected cells were treated with 20 μM MG-132 (Fisher Scientific) and 100 μM leupeptin (Sigma-Aldrich) for 2 h. Cells were isolated in lysis buffer (50 mM Tris, 1 mM ETDA, 0.5% Triton X-100, protease inhibitor (Thermo Scientific), 20 μM 1,10-phenanthroline (Sigma-Aldrich), 20 μM PR-619 (Sigma-Aldrich), 10 mM NEM (Sigma-Aldrich), and 150 mM NaCl in water). Twenty micromolar MG-132 and hundred micromolar leupeptin was added to 1.5-2 mg protein. Primary V5 antibody (Invitrogen, R960-25, 1:500 dilution) was added and samples rocked at RT for 1 h. Subsequently, we added protein A/G magnetic beads (Thermo Scientific) and rocked further at RT for 1 h. Samples were rinsed with wash buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100 in water) twice. 1× Laemmli buffer (Bio-Rad) was added to the beads and heated at 95 °C for 5 min. Beads were magnetically sorted prior to immunoblotting. For his pull-downs, cells were isolated in lysis buffer and His-tag proteins were isolated utilizing Dynabeads His-tag Isolation and Pull-down (Invitrogen) per manufacturer’s protocol.