Figure 6.

CD300b regulates macrophage-mediated phagocytosis of apoptotic cells. (a) J774.1 cells transduced with short hairpin scramble control RNA (shControl), or shRNA targeting CD300b (shCD300b) were stained with Alexa488-labeled anti-CD300b antibody. The efficiency of silencing was assessed by monitoring CD300b cell surface expression via flow cytometry. (b) Control or CD300b-silenced J774.1 cells were mixed with pHrodo-labeled apoptotic cells (AC; 1:4 ratio), and the percentage of J774.1-pHrodo⁺ cells was analyzed by flow cytometry. The contour plots (upper panel) illustrate the gating strategy, whereas the quantification of the percentage of J774.1-pHrodo⁺-cells from three independent experiments is summarized in the bar graph (bottom panel; error bars represent S.E.M.); *P<0.05, **P<0.01 (Student's t-test). (c) Control or CD300b-silenced J774.1 cells were incubated with or without apoptotic cells (AC) for the indicated length of time, and the activation of DAP12-dependent signaling pathways was assessed by western blot analysis with anti-pSyk, anti-pAkt, anti-pErk, anti-pPI3K antibodies. Immunoblot using anti-Syk, anti-Erk, anti-Akt and anti-PI3K-antibodies served as loading controls