Cgp 12177

Acebutolol, alprenolol, atenolol, bucindolol, carazolol, carvedilol, CGP12177, ICI 118551, isoproterenol, labetalol, metoprolol, nadolol, nebivolol, pronethalol, timolol, and 2-{[β-(4-Hydroxyphenyl)ethyl]aminomethyl}-1-tetralone hydrochloride (HEAT HCl) were purchased from Tocris (Bristol, United Kindom). Propranolol HCl and 4-hydroxycarbazole were obtained from Sigma-Aldrich (St. Louis, MO) and bupranolol was obtained from Abcam (Cambridge, UK). All compounds were dissolved in DMSO to obtain a 10 mM stock concentration, which was stored at -20°C. EGF was purchased from Peprotech (Rocky Hill, NJ) and dissolved in sterile deionized water at a 10 ug/mL stock and stored at -80°C. Primers were purchased from IDT (Coralville, IA). The lentiviral vector pLV-H1-EF1α-puro, annealing buffer, packaging vector, and polybrene were purchased from Biosettia Inc. (San Diego, CA). 4-hydroxycarbazole (4-OHC) was purchased from Sigma Aldrich

[³H]-adenine, [³H]-CGP 12177, [¹⁴C]-cAMP, Microscint-20 and Ultima Gold Scintillation fluid were from Perkin Elmer (Coventry, West Midlands, UK). CGP 12177, CGP 20712A, cimaterol and ICI 118551 were from Tocris Bioscience (Bristol, UK). Decyl-β-D-maltopyranoside was from Anatrace (Berkshire, UK). Purified β₁-m23-StaR [8] (link) was provided by Heptares Therapeutics. The following antibodies were used: goat-anti-mouse-Rhodamine secondary antibody (Molecular Probes, Life Technologies, Paisley, UK), horse radish peroxidase-conjugated secondary antibody (Cell Signalling Technology, Leiden, The Netherlands). BODIPY-TMR-CGP (CGP-12177-TMR) was purchased from Molecular Probes (Eugene, OR, USA). Fugene HD transfection reagent and furimazine were from Promega (Southampton, UK). Purified turkey and human ECL2 peptides were obtained from Cambridge Research Biochemicals (Cambridge, UK). All other reagents were from Sigma-Aldrich (Gillingham, UK).

Live cell imaging was carried out using Yokagawa CSU22 spinning disk confocal microscope with a × 100, 1.4 numerical aperture, oil objective and a CO2°C and 37°C temperature-controlled incubator. A 488 nm argon laser and a 568 nm argon/krypton laser (Melles Griot) were used as light sources for imaging EGFP and Flag signals, respectively. Cells expressing both Flag-tagged receptor and the indicated nanobody–EGFP were plated onto glass coverslips. Receptors were surface labelled by addition of M1 anti-Flag antibody (1:1000, Sigma) conjugated to Alexa 555 (A10470, Invitrogen) to the media for 30 min, as described previously. Indicated agonist (isoprenaline, Sigma) or antagonist (CGP-12177, Tocris) (alprenolol, Sigma) were added and cells were imaged every 3 s for 20 min in DMEM without phenol red supplemented with 30 mM HEPES, pH 7.4 (UCSF Cell Culture Facility). Time-lapse images were acquired with a Cascade II EM charge-coupled-device (CCD) camera (Photometrics) driven by Micro-Manager 1.4 (http://www.micro-manager.org).