FIGURE 5.
Effect of HSP90 inhibitor treatment in total and surface levels of N-terminally tagged A2A receptors. A, HEK293 cells stably expressing the tagged receptor FS2-N-A2AR (2 × 105 cells) were incubated with increasing concentrations of the HSP90 inhibitor radiciol or 17-DMAG for 24 h, followed by a 30-min incubation with the radioligand [3H]ZM241385 (1 nm) in the absence and presence of 10 μm XAC. After two washes, surface-bound antagonist was released by acid strip to determine the level of receptors at the cell surface. Error bars, S.D. Data are from four independent experiments done in triplicate. Statistical significance was calculated by analysis of variance followed by Dunnett's multiple-comparison post hoc test (*, p < 0.05; ***, p < 0.001). B, HEK293 cells stably expressing the tagged version G2S-N-A2AR were incubated with increasing concentrations of radicicol or 17-DMAG. After 24 h, cell membranes were prepared, and aliquots (corresponding to 2.5 × 105 cells) were resolved by denaturing electrophoresis and immunoblotted for HSP90α (top), the A2A receptor (middle), and α-tubulin (bottom; loading control).