Cellular uptake and biologic activity of PID-Tat protein in human neutrophils. (A) Neutrophils (2 × 105 cells) were incubated with 1 μM FITC-labeled PID-Tat, and cellular uptake into live cells was analyzed by confocal microscopy as described in “Materials and methods.” (B) Neutrophils (5 × 105 cells) were incubated with 0.5 or 1 μM PID-Tat, followed by centrifugation. The supernatants (S) were collected, the cells were washed with PBS and the wash was collected (W), and the cell pellets were lysed in sample buffer (L). Samples were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-Pak antibody. (C) Neutrophils (2 × 105 cells) were preincubated with GFP-Tat or PID-Tat and then stimulated with 1 μM fMLF for 1 or 5 minutes. Cell lysates of stimulated or unstimulated cells were subjected to SDS-PAGE and probed with anti–phospho Pak antibody (top panel) followed by anti-Pak antibody (bottom panel).
