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.
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