Fig. 4.

Optimization of pharmacochaperone assay in 1,536 well plates. (A) Cell-seeding density: U-2 OS-ADRB2(W158A) (black bars) were seeded at the indicated densities and exposed to control compounds for 16 h. (B) DMSO tolerance: U-2 OS-ADRB2(W158A) cells were seeded at 1,500 cells/well and allowed to attach for 4 h. Cells were incubated in the presence of bortezomib (100 nM) and the corresponding amounts of DMSO indicated as final percent v/v for 16 h. (C) Bortezomib concentration–response curve, each dose run in triplicate. (D) Procaterol and propranolol in the EFC trafficking assay: U-2 OS-ADRB2(W158A)-PK were incubated in the presence of various concentrations (11-point curve) of propranolol or procaterol, each dose run in quadruplicate, for 16 h at 37°C. Note, the Y-axis here is plotted as raw luminescence units rather than normalized percent activity (as in C). This is to better illustrate the higher maximal response elicited by procaterol versus propranolol. The lowest dose in the curve corresponds to vehicle only. Data plotted are mean ± SEM of triplicate data points from at least two independent experiments. Curves represent the best fit of a four-parameter logistic generated using GraphPad Prism 7. DMSO, dimethylsulfoxide.