Proxiplate

The AlphaScreen® assay (Perkin Elmer) was generally performed as previously described.^{55 (link)} In brief, compound plates (1 μL at 10 mM highest concentration; 3-fold, 10-point dilutions in DMSO) were diluted in 1× assay buffer (20 mM TRIS pH 8.0, 25 mM NaCl, 2 mM DTT and 0.05% Tween-20) to 1 mM using a Multimek robotic pipettor (Nanoscreen) and 1 μL was spotted into the wells of 384-well low-volume Proxiplates (Perkin Elmer). To these plates 9 μL of protein-peptide mix in 1× assay buffer was added by Multidrop (Thermo) to bring the final compound concentration to 100 μM and incubated for 30 min at room temperature. Next, 2 μL of a 1:1 mixture of streptavidin-conjugate donor and nickel-chelate or α-GST acceptor beads (45 μg/mL in 1× assay buffer) were added and the plates were allowed to incubate for an additional 30 min in the dark at room temperature. After incubation, the plates were read on an EnVision multi-label reader equipped with an HTS AlphaScreen laser (Perkin Elmer). The expression and purification of the constructs used in this assay was described previously^{56 (link)}.

The AlphaScreen assay was generally performed as previously described.(37 (link)) In brief, compound plates (1 µL at 10 mM highest concentration; 3-fold, 10-point dilutions in DMSO) were diluted in 1× assay buffer (20 mM TRIS pH 8.0, 25 mM NaCl, 2 mM DTT and 0.05% Tween-20) to 1 mM using a Multimek robotic pipettor (Nanoscreen) and 1 µL was spotted into the wells of 384-well low-volume Proxiplates (PerkinElmer). To these plates 9 µL of protein-peptide mix in 1× assay buffer was added by Multidrop (Thermo) to bring the final compound concentration to 100 µM and incubated for 30 min at room temperature. Next, 2 µL of a 1:1 mixture of streptavidin-conjugate donor and nickel-chelate acceptor beads (45 µg/mL in 1× assay buffer) were added and the plates were allowed to incubate for an additional 30 min in the dark at room temperature. After incubation, the plates were read on an EnVision multi-label reader equipped with an HTS AlphaScreen laser (Perkin Elmer). The IC₅₀ values reported are the average of at least 3 values ± the standard deviation. When IC₅₀ values for a single compound were not all active (< 100 µM) or inactive (> 100 µM), the IC₅₀ values were calculated using 4-paramter curve fitting (GraphPad Prism 5) from replicate runs using averaged response values for each compound concentration.

The FRET-assay was performed in white 384-well ProxiPlates (PerkinElmer). Reagents were diluted in assay buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35). Final assay volume (10 μL) contains 3 nM GST-HDAC10 (Life Technologies), 30 nM Tubastatin-Alexa647-Tracer^{[32 (link)]}, and 0.5 nM LanthaScreen Eu-anti-GST (Life Technologies). Compound stocks (10 mM DMSO) were diluted in assay buffer. An 11-fold 1:3-serial dilution of test compounds (1 μL) were presented in the plate and complemented by 9 μL of assay mix. After incubation (1 h, room temperature) TR-FRET was measured with EnVision plate reader (Ex: 3 flashes of the TRF-europiumlaser; Em: 620 and 665 nm (665/620 nm ratio). Inhibition was calculated by using negative control (2 % DMSO) and positive control (20 μM vorinostat). Dose-response curves were fitted in ActivityBase (IDBS) using a four-parameter logistic model and IC₅₀-values were calculated.^{[32 (link)]}

Cyclosporine (cyclosporin A, Enzo Life Sciences), FK506 (tacrolimus, Enzo Life Sciences), rapamycin (Santa Cruz Biotechnology) and proINDY (Tocris Bioscience) were diluted in egg water from 1000x stocks dissolved in dimethyl sulfoxide (DMSO). DMSO (1 μl/ml DMSO) was added to the single treatments and the corresponding DMSO concentration (2 μl/ml) was used as a vehicle control. Larvae were exposed at 5 dpf to treatment solutions or DMSO for a total of 6 hours. Larvae were first treated in a Petri dish for 2 hours, transferred with the treatment solution to white 96-well ProxiPlates (PerkinElmer, 6006290) for 1 hour, and then imaged in the treatment solution for 3 hours. Immediately after exposure, larvae from each treatment group were washed in egg water and transferred to Petri dishes with 50 mL egg water. Larvae that were imaged again at 6 and 7 dpf were given food twice prior to each re-imaging session.

The AlphaScreen® assay was generally performed as previously described.¹⁹ In brief, compound plates (1 µL at 10 mM highest concentration; 3-fold, 10-point dilutions in DMSO) were diluted in 1× assay buffer (20 mM TRIS pH 7.5, 75 mM NaCl, 2 mM DTT and 0.05% Tween-20) to 1 mM using a Multimek robotic pipettor (Nanoscreen) and 1 µL was spotted into the wells of 384-well low-volume Proxiplates (PerkinElmer). To these plates 9 µL of protein-peptide mix in 1× assay buffer was added by Multidrop (Thermo) to bring the final compound concentration to 100 µM and incubated for 30 min at room temperature. Next, 2 µL of a 1:1 mixture of streptavidin-conjugate donor and nickel-chelate or α-GST acceptor beads (45 µg/mL in 1× assay buffer) were added and the plates were allowed to incubate for an additional 30 min in the dark at room temperature. After incubation, the plates were read on an EnVision multi-label reader equipped with an HTS AlphaScreen laser (Perkin Elmer). Dose-response curves were fit using a 4-parameter or 3-parameter fixed top binding equation.