Colocalization of band 3 and 4.2 in Xenopus oocytes. Band-3 (0.5 ng) cRNA alone or B3 with 1.5 ng protein 4.2 or protein-4.2 variant cRNA were injected into Xenopus oocytes; these oocytes were processed for confocal imaging as described in “Materials and methods.” The sections were incubated sequentially with mouse monoclonal anti–band-3 antibody BRIC155, then rabbit anti–protein-4.2 antibody diluted 1:500, and these primary antibodies were detected using a goat antirabbit antibody conjugated to Alexa488 and a goat antimouse antibody conjugated to Alexa546. The micrographs in panels A-C were taken from the same oocyte coexpressing band-3 and 4.2 with (A) band-3 imaged with Alexa546 (red; B), 4.2 Alexa488 fluorescence (green; C), merged image of B3 (A) and 4.2 (B) showing colocalization at the plasma membrane (yellow). The images in panels D-F and H are the corresponding merged images for (D) band-3 and 4.2 Tozeur, (E) band-3 and 4.2 Komatsu, (F) band-3 and 4.2 Nippon, and (H) uninjected oocyte control. The image in panel G shows a section of an oocyte injected with band-3 alone, incubated with BRIC155, and then detected with anAlexa546 secondary. Only protein 4.2 (C) and protein 4.2 Nippon (F) colocalized with B3 at the plasma membrane. Scale bar = 10 μM.