Stock concentrations were determined by UV/VIS spectroscopy using an extinction coefficient of 1,000 M−1 cm−1 at 355 nm for the carboxynitrobenzyl-modified tyrosine (Sreekumar et al., 1998). Selisistat in vivo A 100 μl sample of either 1 mM CYLE or CYD8 in phosphate-buffered saline, pH 7.2, contained in a quartz cuvette, was placed for

30 s in the path of a 100 KHz pulsed q-switched UV laser (DPSS, Santa Clara, Ca) producing ∼800 mW of 354.7 nm light. The samples were then analyzed by RP-HPLC on a 4.6 mm × 150 mm Zorbax SB-C8 column (5 μm particle size) in H2O, MeCN, 0.1% TFA at 1 ml/min. LE and CYLE were resolved by a gradient from 30% to 70% MeCN over 10 min. Dyn-8 and CYD8 were resolved by a gradient from 25% to 70% MeCN over 10 min. Samples were monitored at 210 nm, 280 nm, and 350 nm. Nonphotolyzed CYLE, CYD8, LE, and Dyn-8 samples were prepared and run under the same conditions. Although addition of the parent peptides to photolyzed samples increased the product peaks as expected, further analysis by mass spectrometry confirmed photolytic see more conversion of CYLE to LE and CYD8 to Dyn-8 (data not shown). HEK293 cells stably transfected with a chimeric Gs-Gi protein (S.D. Liberles and L.B. Buck, personal communication; Liberles and

Buck, 2006) were grown in DMEM (Invitrogen) containing 5% FBS (Invitrogen) and 500 μg/ml G-418 (Invitrogen) and maintained at 37°C in an atmosphere of 5% CO2. Cells were plated in 96-well plates at 50,000 cells/well and cotransfected with the GPCR and reporter plasmid using Lipofectamine (Invitrogen) and PLUS reagent (Invitrogen). The transfection media was replaced with ligand-containing DMEM (200 μl/well) and cells were incubated for 36–48 hr 37°C/5% CO2. Care was taken to avoid exposure to bright, nonfiltered light. Each plate was then wrapped in plastic wrap and incubated at 68°C for 2 hr to heat-inactivate native phosphatases. After transferring 100 μl aliquots from each well to a fresh 96-well plate, 100 μl of an

aqueous buffer containing 2 M diethanolamine bicarbonate and 1.2 mM methylumbelliferone phosphate, pH 10.0, was added to each well. Plates were imaged using a Perkin Elmer Envision plate reader using optical settings for methylumbelliferone fluorescence, taking care to image all conditions to be compared on the same day for a single receptor at the same time tuclazepam point after MUP addition. In every individual experiment, the noncaged parent ligand (LE or Dyn-8) was evaluated alongside the caged compound (CYLE or CYD8) at the same receptor and used to normalize data across trials. cDNAs encoding human delta (OPRD1) and human kappa opioid receptors (OPRK1) contained an N-terminal 3xHA epitope tag (Missouri S&T cDNA Resource center, http://www.cdna.org) and the murine mu opioid receptor (OPRM1) contained an N-terminal FLAG epitope tag. Data shown reflect the average of six replicates run in parallel for each condition.