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

Figure 7.

Figure 7

SHP-1 Is More Often Recruited to and Phosphorylated at Larger KIR2DL1 Clusters

(A–M) NKL/KIR2DL1-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-SHP-1 mAb (AF568; A–H) or anti-SHP-1 pY536 Ab (AF568; I–M) and imaged with STED microscopy. (A and I) Mean SHP-1 (A) or SHP-1 pY536 (I) fluorescence intensity in NKL/KIR2DL1-HA cells on isotype control-coated and anti-KIR2DL/S1 mAb-coated slides. (B and J) 3 × 3 μm regions of cell membrane with KIR2DL1 (green) and SHP-1 (B; red) or SHP-1 pY536 (J; red) with overlapping pixels marked in white (left) or with KIR2DL1 clusters color-coded according to their size (right). Each size bin corresponds to one-third of all KIR2DL1 clusters. (C and F) The fractions of all KIR2DL1 clusters associated with SHP-1 that fall into each size group, in cells on isotype control (C) or anti-KIR2DL/S1 (F) mAb. (D and G) The fractions of all KIR2DL1 clusters associated with SHP-1 that fall into each size group in simulated datasets where the positions of KIR2DL1 clusters were maintained and positions of SHP-1 clusters were randomized within the boundaries of cell area, in cells on isotype control (D) or anti-KIR2DL/S1 (G) mAb. (E and H) Average intensity of SHP-1 clusters found nearby KIR2DL1 clusters from specified size bins in cells on isotype control (E) or anti-KIR2DL/S1 (H) mAb. (K) The fraction of all KIR2DL1 clusters associated with SHP-1 pY536 that fall into each size group in cells on anti-KIR2DL/S1 mAb. (L) The fraction of all KIR2DL1 clusters associated with SHP-1 pY536 that fall into each size group in simulated datasets where the positions of KIR2DL1 clusters were maintained and positions of SHP-1 pY536 clusters were randomized within the boundaries of cell area. (M) Average intensity of SHP-1 pY536 clusters found nearby KIR2DL1 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 14–22 cells from two independent experiments (A and I) or 27–33 cells from three independent experiments (C–H and K–M). AU, arbitrary units. ∗p < 0.05, ∗∗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.