Figure 2
From: β-Arrestin1 regulates γ-secretase complex assembly and modulates amyloid-β pathology
β-Arrestin1 enhances γ-secretase activity via its interaction with APH-1. (A) β-arrestin1 directly interacts with APH1-AL. HA-tagged PS1-NTF, PS1-CTF, NCT, APH1-AL and PEN-2 were immunopurified from HEK293T cells with anti-HA resins. Equal amount of 35S-labeled β-arrestin1 protein was incubated with the immobilized proteins (as shown in the lower panel). The bound β-arrestin1 was detected by autoradiography (upper panel). (B) β-arrestin1 interacts with APH1-AL but not other γ-secretase components. Triton X-100 extracts of HEK293T cells overexpressing Flag-tagged PS1-FL, PS1-NTF, NCT, APH1-AL or PEN-2 along with HA-tagged β-arrestin1 were subjected to Co-IP assay using anti-Flag resins. (C) Immunofluorescence analysis showed the β-arrestin1/APH1-AL colocalization. HEK293 cells were stained with FITC-conjugated anti-HA antibody and Cy3-conjugated anti-Flag antibody to probe overexpressed HA-tagged APH1-AL (green) and Flag-tagged β-arrestin1 (red). Scale bar represents 20 μm. (D) Co-IP shows the interaction between β-arrestin1 and APH-1 isoforms. β-Arrestin1 interacts with APH1-AL and APH1-B, but not APH1-AS. (E, F) APH1-AL (E) and APH1-B (F) interacts with wild-type (WT) β-arrestin1 but not the β-arrestin1 8M point mutant. Triton X-100 extracts of HEK293T cells overexpressing βGal, HA-tagged β-arrestin1 WT, β-arrestin1 7M (256-261 6Ala) or β-arrestin1 8 M (282-285 4Ala) along with Flag-tagged APH-1 were subjected to Co-IP. (G) C99 assay. Equal amount of microsomal membrane fractions of HEK293T cells overexpressing βGal, β-arrestin1 WT or β-arrestin1 8M point mutant was subjected to in vitro γ-secretase assay. γ-secretase activity was reflected by production of AICD. (H) Notch processing is not affected by overexpression of β-arrestin1 or β-arrestin1 8M. Lysates of HEK293 cells overexpressing βGal, HA-tagged β-arrestin1 WT or HA-tagged β-arrestin1 8M along with Myc-tagged NotchΔE were subjected to SDS-PAGE, immunoblotted by the indicated antibodies. The generation of NICD represented the Notch-specific γ-secretase activity.
