FcγR engagement causes redistribution of BLT1 and FcγRI receptors to LRs. (A) Detergent-free fractionation of membranes into LR and non-LR fractions. A total of 100 000g membrane fractions from resting AMs was subjected to discontinuous Optiprep gradient centrifugation, as described in “Lipid raft fractionation.” Fractions were harvested and subjected to immunoblot analysis for the LR marker flotillin-1 and the non-LR marker CD45. (B) AMs were pretreated with or without 100 nM LTB₄ for 5 minutes, followed by 10-minute incubation with or without 30:1 IgG-RBCs. Membrane fractions were prepared as in panel A, and the LR (fractions 3-6) and non-LR (fractions 7-10) fractions were pooled and subjected to immunoblot analysis for the proteins of interest shown on the right. The experiment depicted is representative of 3 to 5 independent experiments. (C) Relative microdomain distribution of BLT1, FcγRI, PKC-α, and PKC-δ was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in panel B, and expressed as the LR/non-LR ratio. *P < .05 vs control; #P < .001 vs IgG-RBC alone. (D) AMs were pretreated with 100 nM LTB₄ alone for 5 minutes, membrane fractions were prepared as in panel A, and the LR (fractions 3-6) and non-LR (fractions 7-10) fractions were pooled and subjected to immunoblot analysis for the proteins of interest shown on the right. The experiment depicted is representative of 2 independent experiments. (E) AMs were incubated with (ii,iv) or without (i,iii) 10:1 IgG-RBCs for 10 minutes, and LR patches were stained with CTxB-Alexa 455/anti-CTxB. Anti-CTxB–patched cells were fixed and stained with anti-FcγRI (top) and anti-BLT1 (bottom) antibodies and visualized by confocal microcopy. Images are from one experiment representative of 3 independent experiments.