Alphascreen assay

The AlphaScreen assay (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) was carried out using a 384-well Optiwell microtiter plate as described previously [30 (link)]. Briefly, 1 μL of the translation solution containing biotinylated proteins was added to a well with 14 μL of buffer containing 0.025 μL of serum. The mixture solution at a final concentration of 100 mM Tris-HCl (pH 8.0), 0.01% (v/v) Tween 20 and 0.1% (w/v) bovine serum albumin (BSA), was incubated at 26°C for 30 min. Then, 10 μL of a mixture of streptavidin-coated donor beads and protein G-conjugated acceptor beads (PerkinElmer Life and Analytical Sciences) was added, and incubated at 26°C for 1 h in a dark box. The final concentration of the beads was 12 μg/mL per well. Fluorescence emission at 520–620 nm was measured on the EnVision plate reader (PerkinElmer Life and Analytical Sciences), and the resulting data were analyzed with the AlphaScreen detection program. All repetitive mechanical procedures were performed on a Biomek FX robotic workstation (Beckman Coulter, Fullerton, CA, USA). Translation solution reacted without template RNA served as a negative control. Signals in the negative control well were regarded as noise, and the signal/noise ratio was calculated.

The AlphaScreen assay was performed according to the manufacturer’s general protocol (Perkin Elmer, Rodgau, Germany). Reactions were performed in a 25 μL final volume in white 384-well microtiter plates (Greiner, Frickenhausen, Germany). His-tagged ABL1 (200 nM) and biotinylated myristoylated-peptide derived from the myristoylated N-terminus of ABL1 (myristoyl-GQQPGKVLGDQRRPSLK-Biotin) (GenScript Biotech, Piscataway, NJ, USA) (400 nM) were preincubated 30 m in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM dithiothreitol, and 10 μM polyglycine in the presence of 1% DMSO (100% binding) or in the presence of different concentrations of compounds crizotinib or GNF2 diluted in DMSO. Subsequently, 5 μL of beads containing nickel chelate-coated acceptor beads and streptavidin-coated donor beads was added and incubated in the dark for 60′ at room temperature; the emission of light from the acceptor beads was measured in an EnVision reader (Perkin Elmer) and analyzed using the EnVision Spotfire® Analyst software (Perkin Elmer).

Ligand-induced activation of MC4R was determined by using the AlphaScreen™ assay (Perkin Elmer Life Science) according to the manufacturer’s protocol and has been described elsewhere.^{65 (link)} In brief, 48 h post transfection, cells were challenged with either α-MSH or NDP-α-MSH (1 µM to 0.1 nM) in stimulation buffer (138 nM NaCl, 6 mM KCl, 1 mM MgCl₂ ∙ 6H₂O, 5.5 mM glucose, 20 mM HEPES, 1 mM CaCl₂ ∙ 2H₂O, 0.1% BSA, pH 7.4) containing 1 mM IBMX for 40 min for at 37 °C and 5% CO₂. Incubation was stopped by freezing cells at −80 °C for 10 min prior to cAMP measurements. The determination of cAMP accumulation was performed following to the manufactures’ instructions (Perkin Elmer Life Science) and measured with a plate reader (Mithras LB 940, Berthold Technologies).

The AlphaScreen assay was described previously and performed according to the manufacturer’s protocol (PerkinElmer, USA). The reaction buffer was 50 mM HEPES pH 7.4, 100 mM NaCl, 0.1% BSA, and 0.05% CHAPS. The whole reaction and screening were performed using OptiPlateTM-384 (PerkinElmer, Waltham, MA, USA).
The compounds, acetylated peptide [SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-biotin] and purified bromodomain protein were added to the OptiPlateTM-384 plate. Then, streptavidin-coated donor beads and anti-GST AlphaScreening acceptor beads were added. The incubation condition was 25 °C for 1 h using a Thermomixer C (Eppendorf, USA). The blocking efficacy (IC₅₀) values were obtained by 8-point titration (from 0.0096 to 32 μM). The IC₅₀ values of inhibitors were evaluated with GraphPad Prism 7 using normalized and inhibition analysis suites.