J. performed on the model 6300 Beckman analyzer, and mass spectrometry was carried out using an Ettan matrix-assisted laser desorption ionization time-of-flight apparatus (Amersham Biosciences, Uppsala, Sweden). expression in HeLa-CD4-LTR–gal cells using a previously explained protocol (18). Chemotaxis. The cell migration assay STAT3-IN-3 was conducted using 24-well Transwell migration chambers with 5-m-pore-size polyvinylpyrrolidone-free polycarbonate filters (Corning) as previously explained (59). Data are expressed as the ratio of directed movement (chemotaxis) to random movement and are representative of the results of three impartial experiments where each point represents the mean of three wells 1 standard deviation. Calcium mobilization. Freshly isolated PBMCs or purified monocytes (2 106/ml) were loaded with 3 M Fluo-4 acetoxymethylester (Fluo-4/AM) and 3 M Fura Red/AM (Molecular Probes, Carlsbad, CA) in loading buffer (Hanks’ balanced salt answer without Ca2+ or Mg2+ [Invitrogen] supplemented with 0.5% [wt/vol] bovine serum albumin [Sigma] and 1 mM probenecid [Molecular Probes, Carlsbad, CA] at pH 7.4) according to the manufacturer’s instructions. Cells were then stained with anti-CD14 conjugated to allophycocyanin (APC) at 4C, washed, and resuspended at a density of 2 106 cells/ml in calcium buffer supplemented with 1 mM probenecid at pH 7.4 and kept on ice until required. In some cases, cells were also incubated with 1 M nimodipine, 100 M TMB-8, or 100 nM FS-2 at this stage. Just before the Ca2+ flux STAT3-IN-3 assay, cells were heated to 37C in a water bath. Changes in dye fluorescence after activation with 50 nM Tat over time at 37C were determined by KPNA3 circulation cytometry (FACScan; Becton Dickinson, Mississauga, Ontario, Canada). Calcium mobilization is usually reported as the ratio of Fluo-4 to Fura Red fluorescence intensity over time as calculated using FCSPress v1.4 software (by Ray Hicks, Circulation Cytometry Laboratory, Department of Medicine, University or college of Cambridge, Cambridge, United Kingdom). Equilibrium competition binding assays. GHOST cells were cultured in high-glucose Dulbecco’s altered Eagle’s medium supplemented with 10% (vol/vol) fetal bovine serum, 500 g/ml G418, 100 g/ml streptomycin, 100 U/ml penicillin, and 100 g/ml hygromycin (all from Invitrogen, Carlsbad, CA). Transfected cell media also contained 1 g/ml puromycin (Mediatech, Herndon, VA). Cells were harvested using Cellstripper (Mediatech), washed twice, and resuspended in binding buffer (RPMI 1640 [Invitrogen] supplemented with 1% [wt/vol] bovine serum albumin and 20 mM HEPES) at 1 104 cells/ml. For 125I-labeled chemokine STAT3-IN-3 binding to whole cells, 100 l/well GHOST cell STAT3-IN-3 suspension was placed in 96-well microtiter plates. A 100-l answer made up of 0.1 nM 125I-labeled chemokines (Perkin Elmer) and increasing concentrations of Tat or buffer was then added to the cells. The radiolabeled ligands used were 125I-CCL2 for CCR2+ cells and 125I-CXCL12 for CXCR4+ cells. Specific activity for each chemokine was 2,200 Ci/mmol, as indicated by the manufacturer. Tat concentration varied between 0.01 nM and 10 M. Cells were incubated at 20C for 40 min with gentle agitation and then harvested onto a filter mat using an automatic cell harvester (Skatron Inc., Sterling, VA) and subjected to liquid scintillation counting in a Beckman LS-6000. Determination of cytokine production. (i) Circulation cytometry. PBMCs (1 106/ml) were incubated in 24-well flat-bottomed plates (Falcon) at 37C, 5% CO2 in a 2:1 mix of AIM-V and Iscove’s media (Invitrogen) supplemented with 1% (vol/vol) fetal calf serum (Invitrogen) and 2 M monensin (Calbiochem, La Jolla, CA) STAT3-IN-3 and stimulated with 50 nM Tat or CCL2. After 4 h of activation, cells were stained with monoclonal antibodies.