Colocalization of membrane components to clusters of PhE-BRIC126–labeled CD47 or to polyclonal-labeled Rh.

(A) Positive colocalization: double labeling of CD47 with a noncompeting monoclonal antibody, 6H9. Higher intensity spots of 6H9 (green) generally coincide with the clusters of CD47 produced by PhE-BRIC126 (red). The overlap image shows yellow colocalized regions. (B) Negative colocalization: uniform labeling of the lipid bilayer with fluorescein-phosphatidylethanolamine (FL-PE). Similar images were obtained with antibody labeling of Band 3 as well as glycophorin C, as quantified in Figure 3. (C) Yellow spots in the colocalization image indicate significant overlap of antibody-labeled RhAG (green) with PhE-BRIC126 (red). (D) A polyclonal antibody against Rh(c and E) also generates clusters to which RhAG colocalizes.