Lamp2 is lost during reticulocyte maturation. (A) Different populations of age-synchronized reticulocytes were obtained by Percoll density gradient centrifugation using blood of anemic rats as described in “Methods.” (Top) Analysis of the Lamp2, TfR, and actin content of the reticulocyte subpopulations (0.5 μL of packed cell volume) was carried out by Western blot after SDS-PAGE and transfer on membrane. F1 to F4 correspond to the Percoll fractions from lower to higher density (ie, from younger to older reticulocyte stages). “Retic” stands for the red cells collected before Percoll gradient, “Erythr.” stands for red cells collected from an untreated animal. (Bottom) Coomassie staining of the PVDF membrane before Western blot. (B) Rat reticulocytes were fractionated as described in “Methods.” The different fractions obtained (cytosol, plasma membrane, endosomes, and exosomes; 18 μg of protein) were loaded on a 10% SDS-PAGE, transferred onto PVDF membrane, and stained by Coomassie blue (bottom), then immunoblotted for the proteins indicated on the right (top).