Effect of αR4 on adhesion of red cell ghosts to laminin. (A) Red cells resealed with 40 μM GST or GST-αR4 were incubated for 1 hour at 37°C on BSA- or laminin-coated 96-well microplates. Phase-contrast images show adherent cells after filling the wells with PBS and floating the microplate upside down for 40 minutes before microscopic observation. Cells were viewed with a Zeiss LSM META 510 Confocal microscope (Zeiss, Thornwood, NY) using a lens at 10×/0.30 EC Plan-Neofluar (Zeiss). Images were collected using the Zeiss Confocal microscope laser and the Laser Scanning microscope LSM 510 version 3.2 software (Zeiss). Images were cropped using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). (B) GST, GST-αR4, or GST-αR5 at 100 μM was introduced into red cells. Adhesion of the resealed cells to immobilized laminin was measured using the gravity-driven reverse suspension assay, described for panel A. Adhesion in the presence of GST was normalized as 1. Note the enhanced adhesion in the presence of αR4 fragment but not αR5 fragment; N = 3. (C) αR4 fragment at indicated concentrations was introduced into red cells. Adhesion was measured as described for panel B. Adhesion in the presence of 20 μM GST-α4 was normalized as 1, and the fold change was plotted against increasing concentrations of GST-α4. Note the progressively enhanced adhesion in the presence of increasing concentrations of αR4 fragment; N = 3.