(A) The graphs give the percentage of cell couples that displayed accumulation of the isolated targeting domains, LAT V3, LAT Vav, and LAT PLCδPH as indicated, with the indicated patterns as in Figure 2B relative to tight cell couple formation for wild type 5C.C7 T cells activated with CH27 B cell APCs (10 µM MCC peptide). 30–57 cell couples from 2 to 3 independent experiments were analyzed per condition, 133 total. (B) Wild type 5C.C7 T cells were transduced to express LAT-GFP and sorted for LAT-GFP positive (LAT-GFP pos) and LAT-GFP negative (LAT-GFP neg) T cells or left non-transduced. Cell extracts were blotted for LAT (green) and α tubulin (red) as a loading control. One representative blot of three is shown. (C) LAT band intensities were quantified and are shown relative to endogenous LAT in the non-transduced cells. The GFP intensity used to sort LAT-GFP-positive 5C.C7 T cells corresponds to 6*105 molecules per cell (Singleton et al., 2009). (D, E) For the quantification of the accumulation of LAT and targeted versions thereof at the T cell:APC interface we applied a computational image quantification as recently described in detail (Roybal et al., 2016). We identified a core region (B) of sensor enrichment as defined as the 10% most fluorescent voxels of the average probability distribution across all cells, for all time points, and for all sensors. Using this core region, we calculated the ratio of the amount of the sensor in the region to the average amount across the whole cell. This was done for all time points either under conditions of full stimulus or costimulation blockade as indicated. Imaging data analyzed are the same as in Figures 2 and 3. (F) Wild type 5C.C7 T cells retrovirally transduced to express the indicated LAT constructs were activated with CH27 B cell APCs and 10 µm MCC in the presence of 10 µg/ml anti-CD80 plus anti-CD86. At the given time points T cell:APC cell extracts were blotted for LAT phosphorylation at Y191. Blots were stripped and reblotted with an anti-alpha tubulin antibody as a loading control. One representative blot of two is shown.