Western blot analyses of apoptosis-related proteins in cultured reticulocytes. In vitro–derived reticulocytes were purified by sedimentation at unit gravity and cultured for 110 hours. At various times, aliquots of cells were harvested, washed in PBS, and the total cells were processed for SDS-PAGE and Western blotting. Each lane was loaded with lysate of 2.5 × 106 cells. (A) Proteins associated with mitochondrial damage and apoptosis. Abbreviations: Cyto C, cytochrome C; Apaf-1, apoptosis protease activating factor-1; Procas 3, procaspase 3; Ter-119, an erythroid-specific protein found on the surface of erythroblasts and mature erythrocytes (included as a positive control that is present in all stages of reticulocyte maturation). (B) Effects of incubation pH on the appearance of Bcl-XL in Western blots of 0-hour reticulocyte lysates. Pelleted, nascent (0-hour) reticulocytes were lysed and incubated at 37°C in hypotonic phosphate buffer with the pH shown prior to separation by SDS-PAGE for Western blotting. For comparison, nonincubated lysates of reticulocytes at 0 hours and 110 hours of culture are shown in the first and last lanes, respectively.
