Figure 3. The SKAP55 SH3 domain is... | Rockefeller University Press

Figure 3.

$Figure 3. The SKAP55 SH3 domain is necessary but not sufficient for stabilization of SLP-76 microclusters. (A) Domain structures of the wild-type (WT), SH3 mutant (W333R, “WR”), and SH3-only SKAP55 chimeras (SH3). (B) mRFP1 expression levels in J14.SY cells transiently transfected with the indicated SKAP55.mRFP1 chimeras (n = 3). (C) Cells from B were stimulated, imaged, and presented as in Fig. 2 B. See Table 1 for microcluster properties and experiment numbers. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. (D) Composite microcluster traces for the conditions examined in C. Numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) SKAP55.3×Flag.mCFP chimeras were expressed in J14.SY cells, immunoprecipitated with anti-Flag, and Western blotted for mCFP or ADAP (n = 4). (F) Fraction of cells scored as displaying unstable contacts (n = 4). Error bars indicate mean ± SEM. From parental J14.SY cells expressing mRFP1: **, P < 0.01. From J14.SY cells expressing SKAP55.WT.mRFP1: ††, P < 0.01.$

The SKAP55 SH3 domain is necessary but not sufficient for stabilization of SLP-76 microclusters. (A) Domain structures of the wild-type (WT), SH3 mutant (W333R, “WR”), and SH3-only SKAP55 chimeras (SH3). (B) mRFP1 expression levels in J14.SY cells transiently transfected with the indicated SKAP55.mRFP1 chimeras (n = 3). (C) Cells from B were stimulated, imaged, and presented as in Fig. 2 B. See Table 1 for microcluster properties and experiment numbers. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. (D) Composite microcluster traces for the conditions examined in C. Numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) SKAP55.3×Flag.mCFP chimeras were expressed in J14.SY cells, immunoprecipitated with anti-Flag, and Western blotted for mCFP or ADAP (n = 4). (F) Fraction of cells scored as displaying unstable contacts (n = 4). Error bars indicate mean ± SEM. From parental J14.SY cells expressing mRFP1: **, P < 0.01. From J14.SY cells expressing SKAP55.WT.mRFP1: ††, P < 0.01.