Anti vsvg antibody p5da

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.

SDS-PAGE/immunoblot analysis was performed as previously described (29 (link), 30 (link)) by using the following antibodies: anti-HA antibody (3F10; Roche); anti-Flag antibody (OctA; Santa Cruz); anti-c-Myc antibody (C3956; Sigma-Aldrich); anti-FIV p27 antibody (PAK3-2C1; Santa Cruz); anti-VSVg antibody (P5DA; Roche); anti-FeLV p24 antibody (PF12J-10A; Santa Cruz); anti-RD-114 virus capsid (CA) antibody (37 (link)); and anti-α-tubulin (TUBA) antibody (DM1A [Sigma-Aldrich] or B-5-1-2 [Covance]). For the virus, 500 μl of the virus solution 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 transfected cells, the cells were lysed with radioimmunoprecipitation assay (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).
To calculate the percentage of feline A3Z3 degradation (see percent degradation data in Fig. 5C and D), the band intensities of the immunoblots of feline A3Z3-HA and TUBA were quantified by using Image J software (http://imagej.nih.gov/ij/), and the following formula was used: percent degradation = 100 − {[A3Z3-HA (+Vif)]/[TUBA (+Vif)]}/{[A3Z3-HA (−Vif)]/[TUBA (−Vif)]} × 100.