CR1 colocalizes and coimmunoprecipitates with FAP-1 in human erythrocytes. (A) Colocalization by fluorescence microscopy. Fixed (top row) or fresh (bottom row) erythrocytes were incubated with anti-CR1 mAb 1F11 followed by AlexaFluor-488 goat anti–mouse Ab. After fixation, quenching, and permeabilization, anti–FAP-1 or control Ab was added, followed by AlexaFluor-594 donkey anti–rabbit Ab. Cells were mounted on a slide and images were recorded using the appropriate filters. Bar represents 5 μm. (B) Colocalization by EM-immunogold method. Fresh erythrocytes were reacted with anti-CR1 mAb and then incubated with 18 nm gold conjugated goat anti–mouse antibody as described in “Methods.” Cells were fixed, embedded, sectioned, and then stained with either control Ab (not shown) or anti–FAP-1 rabbit anti-serum (hPTP1E¹⁷ ) followed by 10 nm gold goat anti-rabbit antibody. (C) Fresh (lane 3) or CR1 cross-linked erythrocytes (anti-CR1 mAb YZ1 + goat anti–mouse IgG Ab (lane 5) were lysed and CR1 precipitated with YZ1 coupled to proteins A + G beads (lane 3) or donkey anti–goat coupled to protein A + G beads (lane 5). Controls included: lane 1, erythrocytes + mIgG1 control Ab + protein A + G beads; lane 2, erythrocytes + protein A + G beads; lane 4, erythrocytes + goat anti–mouse coupled to protein A + G beads. The immunoprecipitates were resolved by SDS-PAGE blotted on nitrocellulose membrane and developed with rabbit anti–FAP-1 antiserum.¹⁷  FAP-1 band (arrow) was precipitated in erythrocytes both before (lane 3) and after (lane 5) CR1 cross-linking. Control lanes 1, 2, and 4 were negative. The experiment was repeated 3 times, twice with these results.