Proxiplate 384 well microplates

Ala-peptide binding to Pirh2 or KLHDC10 was evaluated using the AlphaScreen GST detection Kit (PerkinElmer) proximity-based assay. The two assay components, streptavidin-coated donor beads that associate with biotin-labeled peptide and anti-GST acceptor beads that associate with GST-fused protein, are brought in proximity depending on peptide-protein interaction. Upon excitation of donor beads, energy transfer to acceptor beads takes place that generates a luminescence signal. Experiments were performed in ProxiPlate 384-well microplates (PerkinElmer).
Competition experiments to determine IC₅₀ were performed by titrating unlabeled peptides. All required dilutions of reaction components were made in assay buffer (20 mM Tris pH 7.5 at room temperature, 100 mM NaCl, 0.1% Tween 20, 0.05% BSA). 10 nM biotin-labeled Ala-tail peptide (MDELYKAAAAAA) (Biosyntan) and 1 nM GST-Pirh2 or GST-KLHDC10 were pre-incubated with varying concentrations of competing unlabeled Ala-tail variant peptide (GenScript) for 40 min at RT. Following this incubation, pre-mixed donor and acceptor beads (10 μg/mL each) were added to the reaction mixture, in a total 20 μL, and further incubated for 40 min at RT. AlphaScreen signal was measured using PHERAstar microplate reader (BMG Labtech) at RT. Each reaction was performed in triplicates.

Homogeneous time-resolved fluorescence energy transfer (HTRF) assays were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, Massachusetts, US), based on competitive displacement of an europium chelate-labelled cAMP tracer bound to a specific antibody conjugated to acceptor beads. We first established the optimal cell density for an appropriate fluorescent signal. This was done by measuring the TR-FRET signal determined as a function of forskolin concentration using different cell densities. Forskolin dose-response curves were related to the cAMP standard curve in order to establish which cell density provides a response that covers most of the dynamic range of the cAMP standard curve. Cells were not treated or treated with vehicle or 4 μm of the indicated TAT-fused TM peptides for 4 h at 37°C in an atmosphere of 5% CO2. Cells were then grown (1,000 cells/well in white ProxiPlate 384-well microplates, PerkinElmer) in medium containing 50 μM zardaverine, pretreated at 25°C for 15 min with vehicle or the indicated receptor antagonist, stimulated with agonists for 10 min before adding 20 μM forskolin or vehicle and incubated for an additional 15-min period. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).