Figure 4: Inducible localization of ITK-Y511F and ITK-BTK(SH3) to the T-cell-APC contact site. Jurkat cells transfected with fluorescent protein chimeric constructs of WT-ITK, ITK-Y511F, or ITK-BTK(SH3) or with a construct containing fluorescent protein without ITK (pYC), as a transfection control, were incubated with Raji cells that had been pretreated (+SEE) or not treated (−SEE) with SEE for 3 minutes at 37°C. Cells were then fixed in paraformaldehyde and analyzed using epifluorescence microscopy. (a) Representative experiment displaying localization of ITK as YFP fluorescence intensity (right hand panels) in the presence (top panels) or absence (bottom panels) of SEE. Differential interference contrast images are displayed in the left hand panels for ease of orientation. (b) Results of three replicate experiments, performed, and analyzed as in (a), are displayed as the average Localization Index (±SEM) for each ITK construct calculated as described in Section 2. WT/NS denotes nonstimulated cells transfected with WT-ITK, as negative control. The number of conjugates analyzed in each group are WT, 𝑁 = 5 2 ; Y511F, 𝑁 = 3 7 ; BTK(SH3), 𝑁 = 3 2 ; pYC, 𝑁 = 1 0 , WT/NS; 𝑁 = 1 0 .