SKAP55 is dispensable for β₁ integrin clustering downstream of TCR ligation. (A) J14.SY and JSKAP.SY cells were stimulated on anti-CD3–coated plates and fixed after 10 min. Immunofluorescent images of endogenous β₁ integrin (blue) and endogenous talin (red), and direct images of SLP-76.YFP (green) are shown in greyscale and as a pseudocolored merged image (n = 3). (B and C) Higher-resolution merged images of representative J14.SY (B, left) or JSKAP.SY (C, left) cells prepared as in A. SLP-76, talin, and β₁ integrin microclusters were identified algorithmically and used to derive regions of adjacency (right panels). The pseudocoloring scheme is indicated on the right. (n = 3). Bars, 10 µm. (D) Enrichment of talin, SLP-76, and β₁ clustered areas in distinct domains of the contact (edge, middle, and center), relative to the entire contact. (E) Fraction of each domain of the contact occupied by the indicated regions of adjacency. For D and E, data are shown ±SD (error bars) for six cells acquired in three independent experiments. From parental J14.SY cells (with or without mRFP1): °, P < 0.10; *, P < 0.05; **, P < 0.01. (F) Diagram showing the binding sites of the fluorescent probes used in A–E.