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. 2012 Feb 13;209(2):407–421. doi: 10.1084/jem.20111493

Figure 3.

Figure 3.

SLP-76 tyrosines are required for E-selectin–mediated slow leukocyte rolling and Gαi-independent adhesion. (A–C) The carotid artery of chimeric mice reconstituted with BM from WT (n = 3) or Slp76−/− (n = 3) mice (A), with WT (n = 3), SM (n = 3), or DM (n = 3) mice (B), or with WT (n = 3) or ADAP (n = 3) mice (C) was cannulated with a catheter, which was connected to autoperfused flow chambers. Mean rolling velocity of neutrophils on E-selectin (left) and E-selectin and ICAM-1 (right) is presented as means ± SEM. The wall shear stress in all flow chamber experiments was 5–6 dyn/cm2. #, P < 0.05. (D–F) Mixed chimeric mice were generated by injecting BM cells from LysM-GFP+ WT (WT; D–F) mice and Slp76−/− (Slp76−/−; D), SM (E) and DM mice (E), or ADAP−/− mice (ADAP−/−; F) into lethally irradiated WT mice. Cumulative histogram of rolling velocities of 100 GFP+ (WT) and 100 GFP− leukocytes in inflamed cremaster muscle venules of mixed chimeric mice (n = 4) treated with PTx and a monoclonal blocking P-selectin antibody (RB40.34). The insets show the mean rolling velocity ± SEM. #, P < 0.05. (G–I) Numbers of adherent cells per square millimeter in murine cremaster muscle venules. The cremaster muscle was exteriorized 2 h after intrascrotal injection of 500 ng TNF or after injection of TNF and PTx in chimeric mice reconstituted with BM from WT mice (G–I; n = 3) or Slp76−/− (G; n = 3), SM and DM (H), or ADAP−/− (I) mice. Results are presented as means ± SEM. #, P < 0.05. (J) Mixed chimeric mice were generated by injecting retrovirally transduced hematopoietic stem cells (Slp-76–WT construct, WT-c.; Slp76-R448K construct, R448-c.; empty vector, no-c.) from Slp76−/− mice into lethally irradiated WT mice. Cumulative histogram of rolling velocities of transduced (WT-c., n = 100; R448-c., n = 100; no-c., n = 100) leukocytes in inflamed cremaster muscle venules of mixed chimeric mice (n = 3) treated with PTx and a monoclonal blocking P-selectin antibody. The inset shows the mean rolling velocity ± SEM. #, P < 0.05. (K) Coimmunoprecipitation of SLP-76 and ADAP. BM-derived WT neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 min, and then lysates were prepared followed by immunoprecipitation (IP) with ADAP antibody. Precipitates were immunoblotted (IB) with antibodies to total SLP-76 (top) and total ADAP (bottom).