Figure 6.

Kinetics of agonist-induced binding of USP20 with β₁AR and β₂AR are dramatically different. A, HEK-293 cells stably expressing FLAG-β₁AR were stimulated with 1 μm Iso for the indicated times. Receptors were immunoprecipitated (IP) with anti-FLAG affinity gel, followed by immunoblotting for USP20. HEK-293 cells without FLAG-β₁AR were used as control cells (Mock). Center panel, the amount of receptors detected by FLAG antibody. Bottom panels, total cell lysate immunoblotted for USP20 and β-actin. B, USP20 band in each IP was normalized to the cognate β₁AR band, and the mean values (±S.E.) from three independent experiments were plotted as % maximum signals denoting β₁AR/USP20 association. C and D, same procedures were used as in A and B except that HEK-293 cells that stably express FLAG-β₂AR were used. B and D, n = 3, *, p < 0.05 compared with 0 min (′); one-way ANOVA, Bonferroni's post-test.