Research Paper Volume 11, Issue 16 pp 6134—6152

Figure 1. Inhibition effect of Aβ₄₂ (10 μM) on the proteolytic activity of the Arg/N-end rule pathway. (A) Diagram of the ^fDHFR-Ub^R48-X₆₀₉-PTPRN^f (X = Asp, Arg-Asp) fusion. Co-translational cleavage of the fusion by deubiquitylases produces a test protein X₆₀₉-PTPRN^f and a stable ’reference’ protein ^fDHFR-Ub^R48 at the initially equimolar ratio. (B) Degradation of Asp₆₀₉-PTPRN^f in reticulocyte lysate in the presence or absence of Aβ₄₂. Asp₆₀₉-PTPRN^f was expressed in reticulocyte lysate and co-translationally labeled with ³⁵S-Met for 30 min at 30°C in the presence or absence of Aβ₄₂, followed by a chase, immunoprecipitation with anti-flag M2 antibody, SDS-PAGE, and autoradiography. (C) Same as (B) but with Arg-Asp₆₀₉-PTPRN^f fragment. (D) Quantification of (B). The level of Asp₆₀₉-PTPRN^f was normalized on the level of ^fDHFR-Ub^R48. The level of Asp₆₀₉-PTPRN^f detected immediately after stopping of protein expression in reticulocyte lysate (0 min chase) was taken as 100%. "% remaining" is the level of non-degraded Asp₆₀₉-PTPRN^f at shown time points after stopping of protein expression. The absence of Aβ₄₂ - dark-gray column; the presence of Aβ₄₂ – light-gray column. (E) Quantification of (C). Each value is the mean ± SD of at least three independent experiments; *p < 0.01, **p < 0.001.