Fig. 5. Membrane sorting in macropinosomes. (A,B,D) Cells expressing CRAC-GFP were fixed and processed for total immunofluorescence using different antibodies. (A) Representative pictures of newly formed macropinosomes (green) stained with H36, PM4C4, H72 or H161 antibodies (white). (B) Relative intensity of H36, PM4C4, H72 and H161 staining in newly formed macropinosomes was determined and plotted as described in the legend to Fig. 3. H36 and PM4C4 staining was strongly reduced in newly formed macropinosomes. H72 and H161 staining was not. (C) H72 and H161 proteins found in the macropinosomes originate from the plasma membrane. Cells were incubated with H72 or H161 antibodies for 5 minutes at 4°C, washed and allowed to perform macropinocytosis for 3 minutes. Cells were then fixed, permeabilized and processed for immunofluorescence. (D) H⁺-ATPase (white) was not detected in newly formed macropinosomes (green). (E) The cell surface was biotinylated and cells incubated for 0 or 1 hour. Cells were then lysed, the H36 protein was immunoprecipitated, migrated on a polyacrylamide gel, transferred to nitrocellulose and revealed with the H36 antibody (total H36), or with avidin (biot. H36). A control where no antibody was used for the immunoprecipitation (w/o Ab) confirmed the specificity of the immunoprecipitation. Biotinylated surface H36 was not degraded during macropinocytosis. Bars, 4 µm.