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Fig. 10. Effect of cholesterol depletion and repletion on TfR2-mediated ERK1/2 activation. Top panel, serum-starved K562 cells were incubated with or without 10 mM MBCD for 15 minutes at 37°C before treatment with anti-TfR2 mAb or PMA for 5 minutes at 37°C. One aliquot of MBCD-treated cells was incubated with 1 mM water soluble cholesterol. Cells were then collected and lysed. Equal aliquots of total cell lysates from untreated (NT), MBCD-treated (MBCD), and MBCD and cholesterol-treated (MBCD+cholest) cells were resolved by SDS-PAGE and immunoblotted against phospho-ERK1/2. Blots were stripped and reprobed with anti-tubulin Ab to ensure equal protein loading and transfer. Bottom panel, three experiments of this type were evaluated by densitometry to quantify the relative abundance of pERK1/2 induced in response to anti-TfR2 and PMA, normalized with respect to tubulin content and expressed as a percentage of the control (C). Error bars correspond to the range of values obtained in three independent experiments.
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