Figure 5.
Aβ1–40 peptides interact with ABCG2, and overexpression of cloned ABCG2 in cells. A, ABCG2 coimmunoprecipitated with Aβ1–40 peptides. Synthetic Aβ1–40 peptides (5 μm) were mixed with 1 mg of human brain lysates and then incubated with anti-Aβ 6E10 antibody. The precipitated complex were resolved on a 10% SDS-PAGE and probed with an ABCG2 antibody. Lane 1 is human brain lysate incubated with Aβ1–40 peptide and 6E10 antibody. Lane 2 is human brain lysate with Aβ1–40 peptide but not with the antibody 6E10. Lane 3 is human brain lysate after immunoprecipitated (IP) supernatant of lane 1. The co-IP demonstrates that ABCG2 in human brain interacted and coimmunoprecipitated with Aβ1–40 peptides. B, Vector map for pTT5SH8Q2 and cloning site for human ABCG2 cDNA. C, 1–4 were immunocytochemistry showing that ABCG2 protein was localized to the cell membrane of transfected cells (20×, 40×). 5 and 6 show that ABCG2 was not expressed in EGFP vector transfected cells. D, Western blot for ABCG2 protein shows that both the monomer at 72 kDa and a high-weight posttranslation-modified forms of ABCG2 were expressed in transfected HEK293 cells at 24 h after transfection (lanes 2 and 4) and highly expressed at 48 h (lanes 6 and 8). ABCG2 was not expressed in the empty vector-transfected controls (lanes 1, 3, 5, and 7) at 24 and 48 h after transfection. Lane 1, Empty vector 24 h, clone 1. Lane 2, ABCG2 vector 24 h, clone 1. Lane 3, Empty vector 24 h, clone 2. Lane 4, ABCG2 vector 24 h, clone 2. Lane 5, Empty vector 48 h, clone 1. Lane 6, ABCG2 vector 48 h, clone 1. Lane 7, Empty vector 48 h, clone 2. Lane 8, ABCG2 vector 48 h, clone 2.