Fig. 1. Association of TfR2 with lipid rafts/caveolae by density gradient centrifugation. (A) K562 cav, K562 wt and HepG2 cells were lysed in Triton X-100 and subjected to sucrose gradient centrifugation. Aliquots of fractions collected from the top of the gradient were analyzed by western blotting with the antibodies against TfR2, CD81, TfR1 and caveolin-1. Equal protein amounts for each fraction were resolved by SDS-PAGE. (B) HepG2 cells were serum-starved overnight and subjected to sucrose gradient centrifugation (top panels). In a second set of experiments, serum-starved HepG2 cells were pre-treated with human holotransferrin (4 hours at 37°C), before sucrose gradient and western blot analysis.