Detection of EAAT3 using Western blot and indirect immunofluorescence. (A-B) Western blots of the membrane fraction of uninfected human erythrocytes. For panel A approximately 2 × 107 cells were lysed and the membrane fraction was separated on a 5%-15% SDS-PAGE. The separated membrane proteins were blotted onto nitrocellulose and probed with a monoclonal antibody against EAAT3 (left lane). Spectrin served as a loading control. Incubation of the blot with the secondary (rabbit anti–mouse) antibody alone gave no detectable bands (right lane). For panel B approximately 5 × 107 cells were lysed and the membrane fraction was separated on a 4%-12% SDS-PAGE. The gel was processed as described for panel A. (C) IFA images showing punctate staining of the membrane of both uninfected (RBC) and P falciparum–infected (iRBC) human erythrocytes by the anti-EAAT3 monoclonal antibody. Uninfected erythrocytes (left panel) and erythrocytes infected with trophozoite stage parasites (right panel) were fixed and incubated either without antibody, with the secondary antibody alone, with an antibody against human band 3, or with an anti-EAAT3 monoclonal antibody. The cells were subsequently incubated with an anti–mouse antibody conjugated to the fluorescent dye Cy3 and analyzed by fluorescence microscopy.
