Fig. 4.

TCS effects on MARCKS translocation to the cytosol in Ag-stimulated RBL-2H3 cells. RBL cells were transfected with the mRFP-MARCKS-ED 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, 5 μm. As described in Methods, the percentage of transfected cells with MARCKS that translocated to the cytosol 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= 80 for Ag-only cells and N=45 for Ag + 10 μM TCS cells. Percentage translocation and percentage ruffling values in (B) and (D) are from three independent days of imaging, ± SEM. Translocation time values in (C) are averages over the 80 (Ag) and 45 (Ag + 10 μM TCS) cells, ± SEM. Statistically significant results, comparing Ag-stimulated cells ± TCS exposure, are represented by ** p<0.01, determined by one-tailed t-test.