Figure 1

β-Arrestin1 modulates Aβ production and γ-secretase activity. (A) β-arrestin1 modulates endogenous Aβ production in non-AD mice. Cortical (Cx) and hippocampal (Hp) levels of Aβ40 and Aβ42 in wild-type (WT) and β-arrestin1-knockout (βarr1^−/−) mice (n = 4-5 for each group) were measured by ELISA. (B) Full-length APP and APP fragments in the hippocampal extracts from β-arrestin1-knockout mice. Protein extracts were subjected to western blot using antibodies against APP and actin. (C) Activities of α-, β-, and γ-secretase in hippocampal extracts of WT and βarr1^−/− mice were measured by specific fluorogenic substrate assay (n = 4 for each group). Data are shown as mean ± SEM of at least three independent experiments. (D) Mouse primary neurons were transfected with β-arrestin1 specific siRNA or non-specific control siRNA and were cultured for 7 days (upper panel). Secreted Aβ levels (middle panel) were measured by ELISA and γ-secretase activity (lower panel) was measured by specific fluorogenic substrate assay. (E) Mouse primary neurons were transfected with vectors carrying HA-tagged human β-arrestin1 cDNA or control vectors (upper panel). 48 h after transfection, secreted Aβ levels and γ-secretase activity were measured as (D). Data are shown as mean ± SEM of at least three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. NS, non-specific oligomer; Ctrl, control. (F) C99 γ-secretase assay. Equal amount of microsomal membrane fractions of HEK293T cells overexpressing βGal or human β-arrestin1 was subjected to in vitro γ-secretase assay using purified C99. γ-secretase activity was reflected by production of AICDs. Specificity of the reaction was validated by γ-secretase specific inhibitor L-685,458 (10 μM, Calbiochem).