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. 2016 May 19;15(9):1957–1972. doi: 10.1016/j.celrep.2016.04.075

Figure 6.

Figure 6

ZAP-70 Is More Often Recruited to and Phosphorylated at Larger KIR2DS1 Clusters

(A–M) NKL/KIR2DS1-HA cells were incubated for 5 min on isotype control-coated or anti-KIR2DL/S1 mAb-coated slides, fixed, stained with anti-HA mAb (AF488) and anti-ZAP-70 Ab (AF568; A–H) or anti-ZAP-70 pY319 Ab (AF568; I–M) and imaged with STED microscopy. (A and I) Mean ZAP-70 (A) or ZAP-70 pY319 (I) fluorescence intensity in NKL/KIR2DS1-HA cells on isotype control-coated and anti-KIR2DL/S1 mAb-coated slides. (B and J) 3 × 3 μm regions of cell membrane with KIR2DS1 (green) and ZAP-70 (B) or ZAP-70 pY319 (J; red) with overlapping pixels marked in white (left) or with KIR2DS1 clusters color-coded according to size (right). Each size bin corresponds to one-third of all KIR2DS1 clusters. (C and F) The fractions of all KIR2DS1 clusters associated with ZAP-70 that fall into each size group in cells on isotype control (C) or anti-KIR2DL/S1 (F) coated slides. (D and G) The fractions of all KIR2DS1 clusters associated with ZAP-70 that fall into each size group in simulated data where the positions of KIR2DS1 clusters were maintained and positions of ZAP-70 clusters were randomized within the boundaries of cell area, in cells on isotype control (D) or anti-KIR2DL/S1 (G) coated slides. (E and H) Average intensity of ZAP-70 clusters found nearby KIR2DS1 clusters from specified size bins in cells on isotype control (E) or anti-KIR2DL/S1 (H) mAb-coated slides. (K) The fraction of all KIR2DS1 clusters associated with ZAP-70 pY319 that fall into each size group, in cells on anti-KIR2DL/S1 mAb. (L) The fraction of all KIR2DS1 clusters associated with ZAP-70 pY319 that fall into each size group where ZAP-70 pY319 clusters were randomized within the boundaries of cell area. (M) Average intensity of ZAP-70 pY319 clusters found nearby KIR2DS1 clusters from each size bin. (A and I) Each symbol represents one cell. Horizontal bars and errors represent the medians and interquartile range. (C–H and K–M) Bars and errors represent the medians and interquartile range. The data are from 13–18 cells from two independent experiments (A and I) or 29–39 cells from three independent experiments (C–H and K–M). AU, arbitrary units. ∗∗p < 0.01,∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Mann- Whitney test (A) and (I) or row-matched Friedman test with Dunn’s post-test (C–H and K–M).

See also Figure S7.