Figure 4.

FcγRI and BLT1 colocalize and associate in AM LR microdomains after FcγR engagement. (A) AMs were incubated with (ii) or without (i) 10:1 IgG-RBCs for 10 minutes, and the cells were fixed. They were then stained with anti-BLT1 and anti-FcγRI primary antibodies, followed by fluorescein isothiocyanate-labeled and rhodamine-labeled secondary antibodies, respectively, and visualized by confocal microcopy. Confocal images were taken with identical settings to allow comparison of staining. Images are from one experiment and are representative of 5 independent experiments. (B,D-E) AMs were incubated with or without 30:1 IgG-RBCs for 10 minutes, and membrane fractions were obtained by Optiprep gradient centrifugation. LR and non-LR membrane fractions were pooled, and pooled LR or non-LR fractions were immunoprecipitated with an antibody against either FcγRI (B), BLT1 (D), or nonspecific rabbit IgG (E), as described in “Phagocytosis and bacterial killing.” The immunoprecipitates were subjected to immunoblot analysis using antibodies against the proteins shown on the right. The data shown are from a single experiment representative of 3 independent experiments. (C) Association of BLT1 and Gαi3 with FcγRI was determined by densitometric analysis of immunoblots of FcγRI immunoprecipitates from 3 different experiments, as depicted in panel B, and expressed as the LR/non-LR ratio of each protein. *P < .05 vs control.