Figure 6. Csk localisation does not require PAG^pY314.

(A-B) Ag-experienced F5 (FynWT) and Ag-experienced F5 FynKO CD8⁺ T cells were crosslinked with TCRβ/CD8α and Streptavidin AF543 for 5 min fixed, permeabilised and intracellular proteins were double stained for Csk with Lck and Lck^pY505. (B) The sum of fluorescence above background of each condition was calculated using Volocity software in both the proximal and the distal half of the cell. The data set of 1 experiment, comprising 25 images for each condition was used to generate the protein distribution graph and calculate p-values as determined by the Student’s two-tailed t-test; where p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***, p≤0.001 = ****. The data is representative of 2 independent experiments. (C) Ag-experienced CD8⁺ T cells were treated for 20 min with Na₃VO₄, stained with TCRβ/CD8α, fixed, permeabilised and stained for Csk, with nuclei stained with DAPI. A single 2D optical section (along x-y axis) is shown in each panel and an overlay of red and white pixels is represented as a merge image. Scale bar represents 1.3μM. Arrows indicate distribution of Csk in cytoplasm. Data is representative of at least 50 cells of each condition from 3 independent experiments. (D) Ag.experienced CD8⁺ T cells were conjugated to NP68- or GAG-pulsed RMA-S cells for 5-min (top 2 panels) or activated by TCRβ/CD8α cross-linking (bottom 2 panels) and fluorescently labeled with Abs to Csk and γ-tubulin to label the centrosome. Images were rendered with Volocity software. Representative conjugates containing GAG-pulsed RMA-S (first panel) or NP68-pulsed RMA-S (second panel) each intracellularly labelled with Mitotraker (white) and a single Ag.experienced CD8⁺ T cell. Scale bar represents 6 μM. Data is representative of at least 50 cells of each condition from 2 independent experiments.