FIG. 4.

Vif does not induce degradation of CypA. (A) HeLa cells (5 × 10⁶) were transfected with 0.5 μg of pcDNA-HA-CypA and either 4.5 μg of pNL-A1 DNA (Vif+) or 4.5 μg of pNL-A1vif(−) DNA [Vif(−)]. Cells were harvested 24 h later and pulse-labeled for 10 min with [³⁵S]methionine (2 mCi/ml). Unincorporated isotope was removed, and cells were cultured at 37°C in complete RPMI medium. Aliquots were collected at the indicated times and stored on dry ice. Cells were then lysed in 300 μl of lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.5% Triton X-100). The cell extracts were centrifuged at 13,000 × g for 3 min, and half of each supernatant was immunoprecipitated with an HA-specific rat monoclonal antibody (CypA) or a Vif-specific polyclonal rabbit antiserum (Vif). Immunoprecipitated proteins were separated by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis and visualized by fluorography. (B) CypA-specific bands were quantified by densitometric scanning, and results are plotted as percentages of the CypA signals detected at the pulse time points, which were defined as 100%. (C) Vif-specific bands were quantified as described in the legend to panel B.