A, Human embryonic kidney cells (293-ET) were transfected with an ABCA1 cDNA in the presence or absence of vectors expressing the HIV-1 Nef protein either in the anti-sense orientation (−) or in the sense orientation (+). 24 hours post-transfection, protein lysates were prepared from the cells and the amount of ABCA1, Nef and β-actin in the lysates was determined by immunoblotting (IB). B, Co-expression of Nef with cMIR, another transmembrane protein, in 293ET cells does not effect cMIR protein expression. C, Co-expression of Nef does not inhibit ABCA1 mRNA levels as determined by RT-QPCR assays indicating Nef is down-regulating ABCA1 protein levels by a post-transcriptional mechanism (n=4, ±SEM, p=0.37). The Nef mediated suppression of ABCA1 protein levels also occurs in HEK293 and HeLa cells (D), and is abrogated by the NefG2A mutation, which blocks the myristoylation and membrane association of Nef (E).