Figure 6

Functional characterization of the HA-tagged ABCA1. A. The apoA-I-dependent cholesterol efflux was measured in HEK293H and MDCKII cells expressing the untagged (WT) and HA-tagged (HA-WT) ABCA1. As a control the parental cell lines were used. Western blots at the bottom, developed with anti-ABCA1 antibody, indicate the expression levels in the studied cell lines. ABCA1 expression significantly elevated the apoA-I-specific cholesterol efflux in both cell types. HA-tagging did not alter the function of ABCA1. n.d. - not detected. B. Binding of Cy5-labeled apoA-I was determined in HEK293H cells expressing the untagged (WT), the HA-tagged wild type ABCA1 (HA-WT) and the mutant form of HA-ABCA1 (HA-MM). Substantial labeling was observed in cells expressing the untagged and the HA-tagged wild type ABCA1 (colored histograms) as compared to parental cells (grey histograms). No apoA-I binding was seen in cells expressing the mutant form of HA-ABCA1. 4 h pretreatment with the calpain inhibitor, ALLN (50 μM) significantly increased apoA-I-binding in both untagged and HA-tagged wt ABCA1-expressing cells, whereas 50 μM cylcosporin A (CsA) pretreatment slightly reduced the apoA-I-binding in these cells. ALLN and CSA had no effect on apoAI-binding of the parental and mutant HA-ABCA1 expressing cells. Control - cells treated with vehicle.