Fig. 6. Immunofluorescence analyses of TPA-dependent traffic of PKC. Control (empty-vector-transfected) RBL cells (A-J) and RBL-Syt II^- cells (A'-J') were treated with TPA for the indicated time periods: 0 minutes (A,A'); 20 minutes (B-D,B'-D'); 2 hours (E-G,E'-G'); or 4 hours (H-J,H'-J'). During the last 5 minutes of incubation with TPA, FITC-Tfn (50 µg/ml) was added. The cells were subsequently processed for immunofluorescent staining using rabbit polyclonal anti-PKC antibodies (1:200 dilution) and Rhodamine-conjugated donkey anti-rabbit antibodies. The cells were visualized by confocal microscopy, as described in the Materials and Methods. Red, PKC; green, Tfn; yellow, overlap. Data represent one of six separate experiments.