2030 arvo x multilabel counter instrument

Western blotting and reporter assay were performed as previously described [12 (link)–14 (link), 45 (link)–47 (link), 52 (link)]. For the Western blotting of virus particles, 340 μl of the culture supernatant was ultracentrifuged at 100,000×g for 1 h at 4 °C using a TL-100 instrument (Beckman), and the pellet was lysed with 1 × SDS buffer. For the Western blotting of transfected cells, the cells were lysed with RIPA buffer (50 mM Tris–HCl buffer [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche). The following antibodies for Western blotting: anti-His polyclonal antibody (OGHis; Medical and Biological Laboratories), anti-HA antibody (3F10; Roche), anti-FIV p24 Capsid antibody (PAK3-2C1; Santa Cruz Biotechnology); anti-alpha-tubulin (TUBA) antibody (DM1A; Sigma), and anti-VSVg antibody (P5DA; Roche). For FIV reporter assays, HEK293T cells were used for the target of infection. Ten microliter of the culture supernatant of transfected cells was inoculated into HEK293T cells in a 96-well plate (Nunc), and the firefly luciferase activity was measured by using the BrillianStar-LT assay system (Toyo-b-net) and the 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ procedures.

The culture supernatant harvested at 48 hours post-transfection was centrifuged and filtered through a 0.45-μm filter (Millipore) to produce virus suspensions. The infectivity of the virus suspensions was measured by a TZM-bl assay as previously described50 (link)51 (link). Briefly, 100 μl of the virus solution were inoculated onto TZM-bl cells in a 96-well plate (Nunc), and the β-galactosidase activity was measured using the Galacto-Star mammalian reporter gene assay system (Applied Biosystems) and a 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ instructions. Western blotting was performed as previously described37 (link)50 (link)51 (link) using the following antibodies: anti-CA polyclonal antibody (ViroStat), anti-flag polyclonal antibody (OctA; Santa Cruz Biotechnology), anti-HA antibody (3F10; Roche), and anti-alpha-Tubulin (TUBA) antibody (DM1A; Sigma). The band intensity of bovine A3Z3-HA (Figs 3A,C, 4B,D and 5B,C and Supplementary Figure 5) was quantified using ImageJ software (http://rsbweb.nih.gov/ij/).

The culture supernatant harvested at 2 days posttransfection was centrifuged and then filtered through a 0.45-μm-pore-size filter (Millipore) to produce a virus solution. The infectivity of the virus solution was measured by luciferase assay. Briefly, 100 μl of the virus solution was inoculated into HEK293T cells in a 96-well plate (Nunc), and the firefly luciferase activity was measured by using a BrillianStar-LT assay system (Toyo-b-net) and a 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers' procedures.