Subcellular localization of reticulocyte iPLA2. Rat reticulocytes were fractionated as described in “Reticuloate subcellular fractionation.” The different fractions obtained (plasma membrane [PM], cytosol, endosomes, mitochondria, and exosomes; 100 μg protein) were loaded on a 12% SDS-PAGE and (A) stained by Coomassie blue or (B) immunoblotted for the proteins indicated on the right. (C) Endosomal vesicles were prepared and loaded on a linear sucrose gradient as described in “Sucrose gradient analysis.” Fractions were collected and after TCA precipitation, proteins were separated by SDS-PAGE and analyzed by Western blot for the presence of the proteins indicated on the right. (D) Exosomes aseptically collected from in vitro maturation of rat reticulocytes were loaded on SDS-PAGE and analyzed for the presence of a 30-kDa cleavage product of iPLA2 by Western blot, before (t₀) and after incubation for 48 hours at 37°C. In panels B and D, unfilled arrows indicate the native form of iPLA2 (MW 85 kDa); black arrows indicate the putative 30-kDa cleavage product of iPLA2. The molecular mass (kDa) standards are indicated on the left.