Fig. 1.
Proteasomal degradation of ABCA1 in macrophages. Peritoneal macrophages from C57 mice were incubated with 50 μg/ml acetyl-LDL and 3 μM TO901317 for 24 h to induce ABCA1 expression. The macrophages were incubated with or without 20 μM MG132 for 4 h. Cell lysates were subjected to immunoblotting using Bafilomycin A1 against ABCA1 and vinculin. Vinculin was used as a loading control.
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CSN complex associates with ABCA1
As the first step, to reveal the mechanism and regulation of proteasomal degradation of ABCA1, we analyzed the putative interaction between ABCA1 and CSN, a key molecule in controlling protein ubiquitination and deubiquitination. HEK293 cells were transiently transfected with expression vectors for ABCA1 and Flag-tagged CSN5, a subunit of the CSN complex, and ABCA1 was immunoprecipitated with anti-ABCA1 antibody. As shown in Fig. 2A, Flag-tagged CSN5 was coimmunoprecipitated with ABCA1 in the presence of MG132, but not in its absence. The association of ABCA1 and Flag-tagged CSN5 in the presence of MG132 was confirmed by their coprecipitation with anti-Flag antibody (Fig. 2B).
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Distribution patterns Although both species are considered
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