Figure 3

Human-DPPIV causes a decrease in phosphorylation of HIV1-Tat in Sf9 cells. (A) Lysates of Tat-expressing Sf9 cells harvested at the indicated time were subjected to IP with anti-phospho-serine mAb (upper panel) or anti-Tat mAb (lower panel). Each immunoprecipitate was separated by SDS-PAGE, blotted and subsequently probed for with anti-Tat mAb. The band in lane 4 (lower panel) was cut from another position of the same blot. (B) The intensities of phospho-Tat protein bands were determined with the QuantityOne software and calibrated against the respective intensities of the total proteins. Tat being expressed alone for 72 h was taken as standard (= 100%). Each bar represents the relative phosphorylation level deduced from 3 independent experiments ± standard deviations as a percentage of the standard. (C) Lysates of Tat-expressing Sf9 cells were treated with different concentrations of alkaline phosphatase for 2 h at 37°C as follows: 1 U alkaline phosphatase/500 μg protein (lane 2), 1 U alkaline phosphatase/200 μg protein (lane 3) or 1 U alkaline phosphatase/100 μg protein (lane 4). Control samples were left untreated (lane 1 and lane 5) then immunoprecipitated with the anti-phospho-serine mAb (upper panel) or anti-Tat mAb (lower panel). The immunoprecipitates were blotted and subsequently probed for with anti-Tat mAb.