Fig. 2.

TCS effects on PKC βII translocation to the plasma membrane in Ag-stimulated RBL-2H3 cells. RBL cells were transfected with the YFP-PKC βII-YFP plasmid. Live cell time-lapse images were taken for 10 min immediately after addition of 0.005 μg/ml Ag or 0.005 μg/ml Ag + 10 μM TCS in the cells by confocal fluorescence microscopy. Representative images per group, pre-exposure and post-exposure, immediately following first translocation (at ~170–250 sec post-Ag), are shown in (A). Scale bar, 10 μm. As described in Methods, the percentage of transfected cells with PKC βII that translocated from the cytosol to the plasma membrane within 10 min post addition of Ag ± 10 μM TCS are plotted in (B); first time of this translocation is plotted in (C). As a control showing cellular inhibition by TCS, the percentage of transfected cells undergoing membrane ruffling within 10 min (D) was calculated using Fiji image J. For (B-D), values presented are derived from analysis of N= 43 for Ag-only cells and N=50 for Ag + 10 μM TCS cells. Percentage translocation and percentage ruffling values in (B) and (D) are from six independent days of imaging, ± SEM. Translocation time values in (C) are averages over the 43 (Ag) and 50 (Ag + 10 μM TCS) cells, ± SEM. Statistically significant results, , comparing Ag-stimulated cells ± TCS exposure, are represented by * p<0.05, ** p<0.01, determined by one-tailed t-test.