Alphascreen assay kit

Chemical libraries for HTS were acquired from various sources. An additional batch of compound 1 was purchased from ChemBridge Corp. (San Diego, CA) for revalidation. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Restriction DNA endonucleases and DNA ligases were purchased from New England Biolabs (Beverly, MA). SYBR green I stain was purchased from Life Technologies (Carlsbad, CA). The AlphaScreen assay kit was purchased from PerkinElmer (Waltham, MA). Taq DNA polymerases for PCR, the PCR purification kit, and the Plasmid Miniprep kit were purchased from Qiagen (Valencia, CA). COS7 and HeLa cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium containing 10% FBS. All cells were maintained in an incubator at 37°C in a humidified atmosphere of 5% carbon dioxide.

PDE activity assay was performed as described previously with modification [12 (link), 13 (link)]. Frozen heart tissue samples were homogenized in PDE assay lysis buffer [40 mM TrisHCl · (pH 7.5), 15 mM β-mercaptoethanol, 20% (vol/vol) glycerol, 1 mM Na₃VO₄, 100 nM Okadaic acid, Abcam protease inhibitor mixture]. cAMP/cGMP–PDE activities were measured using 1 μM cAMP or cGMP substrate in the presence of 4μg/mL calmodulin and 0.8 mM CaCl₂ (for Ca²⁺/CaM-dependent PDE assay). Briefly, PDE assay reactions were assembled on ice until addition of cAMP substrate. Reactions were incubated at 25 °C for 10 min and then were terminated at 100 °C for 1 min. After cooling at RT for 10 min, the remaining cAMP or cGMP level in the reaction lysate was measured by AlphaScreen assay kit (PerkinElmer). PDE1 activities were defined as total PDE activity minus PDE activity in the presence of PDE1 inhibitor IC86340 (15 μM). Activities were normalized to protein concentration.

MLAF50 was synthesized by using the method described in Supporting Information. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. All oligonucleotides for plasmid constructions were synthesized by Integrated DNA Technologies (Coralville, IA). The In-Fusion cloning kit (Takara Bio USA, Mountain View, CA) was used according to the manufacturers’ recommendations. The AlphaScreen assay kit was purchased from PerkinElmer (Waltham, MA). The anti-PCNA PC10 mouse IgG was purchased from Cell Signaling Technology (Danvers, MA). U2OS cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium containing 10% FBS. All cells were maintained in an incubator at 37°C in a humidified atmosphere of 5% carbon dioxide.

Direct binding of ligand candidates to DAF-12 was assessed using the Alphascreen assay kit (Perkin Elmer) as described previously (Motola et al., 2006 (link)). Also see Supplemental Experimental Procedures. Supplemental

cAMP assays were performed using the AlphaScreen® Assay Kit (PerkinElmer) as previously described (Chen et al., 2012 ). Briefly, cultured HEK cells stably expressing the α_2AAR (Tan et al., 2002 (link); Cottingham et al., 2011 (link)) were resuspended with the stimulation buffer (1× HBSS, 0.1% BSA, 0.5 mM IBMX, 5 mM HEPES, pH 7.4) and mixed with anti-cAMP acceptor beads. The mix was divided into 3 groups and incubated at 37°C with the following chemicals: (1) vehicle; (2) 10 µM forskolin (Sigma); (3) 10 µM forskolin and ligand of interest. After a 20-min treatment, biotinylated cAMP/streptavidin donor beads (in 0.1% BSA, 0.3% Tween-20, 5 mM HEPES, pH 7.4) were added to the mix and incubated for an additional 30 min at room temperature. Fluorescence intensity was then analyzed on a Biotek Synergy2 plate reader.