Endocytosis and exocytosis of GPA-positive vesicles in maturing reticulocytes. (A) Unfiltered reticulocytes cultured using protocol B were labeled with BRIC256 at 10°C, followed by incubation at 37°C for 0, 10, 20, 40, and 60 minutes. Incubation with rabbit anti–mouse Fab before permeabilization ensured that any external GPA stained red (Alexa Fluor 546) and internal GPA stained green (Alexa Fluor 488). (B) After a BRIC256 (green) internalization assay for 20 and 60 minutes, reticulocytes were dual stained with anti-LC-3 (red). Scale bars indicate 5 μm. (C) Scanning electron microscopy of filtered reticulocytes showing membrane blebs. Scale bars indicate 5 μm. (D) Live-cell imaging of a reticulocyte showing membrane blebbing (left: phase contrast; right: labeled with anti–GPA-FITC). Scale bars indicate 5 μm. (E) Immunoblot of vesicles purified from filtered reticulocytes (i, iii, v, vii, ix, and xi) and from membranes made from these reticulocytes (ii,iv,vi,viii,x,xii) using rabbit anti-glycophorin A (i-ii), mouse monoclonal anti–band 3 (BRIC170; iii-iv), rabbit anti-GLUT1 (v-vi), mouse monoclonal anti–β actin (clone AC-15; vii-viii), rabbit anti-ankyrin (with cross-reactivity for both α and β spectrin; ix-x), and mouse monoclonal anti α spectrin (BRIC174; xi-xii).