Western lightning plus ecl substrate

HEK 293a cells transfected with the appropriate vector (either pcDNA3.1 or PTHR₁) or HOS cells were grown in white 24-well microplates with clear bottom (VisiPlate-24, PerkinElmer, Whaltham, MA, USA). Cells were treated with the CMs for 30 minutes at 37 °C followed by three rinsing with HBSS. After this step, 500 µL of the luminol-based Western Lightning Plus-ECL substrate (PerkinElmer) was added to each well. Using a TECAN Infinite® 200 PRO microplate reader (Tecan Trading AG, Männedorf, Switzerland), luminescence readings were obtained for each well. The colorimetric detection of PTHR₁ bound fusion proteins was performed using TMB in clear 24-well microplates. Briefly, following the incubation with the fusion protein, cells were rinsed three times using HBSS and were further incubated 30 minutes at room temperature with 250 µL of a TMB solution. The reaction was stopped using an equivalent volume of 0.2 M HCl solution turning the blue oxidation product of TMB yellow, and the plate was read for absorbance at 450 nm using a TECAN Infinite® 200 PRO microplate reader.

Zebrafish embryos were lysed in ice-cold lysis buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% Triton X-100) containing 1 x protease inhibitor cocktail (Sigma-Aldrich). The samples were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, probed with anti-myc (1/1000, Santa Cruz Biotechnology) and anti-α-tubulin (1/1000, GeneTex, GTX124303) antibodies, and detected using the Western-Lightning Plus-ECL substrate (PerkinElmer).

Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane (Millipore Sigma, MA). After blocking with 5% skim milk, membrane was incubated overnight at 4 °C with primary antibodies against Erk1/2, phospho-Erk1/2 (Thr202/Tyr204), Akt, phospho-Akt (Ser473) (Cell Signaling Technology, MA) and β-actin (Wako, Japan). Then, membrane was incubated with either anti-mouse or anti-rabbit HRP–conjugated secondary antibodies, washed and visualized with the Western Lightning Plus ECL Substrate (Perkin Elmer, MA).

Whole kidneys were homogenized and protein was extracted from 20 mg of tissue in 1 ml RIPA buffer supplemented with protease inhibitors (Roche) for 2 hrs. 10 μg of protein was separated by SDS-PAGE using a 10% separating and a 4.5% stacking gel and transferred to a PVDF membrane with 0.2 μm pore size (Roth) via a semidry transfer. The membrane was blocked with 5% skim milk in TBS-T, cut in half and probed with the mouse anti-β-catenin (1:1000, BD Transduction Laboratories, 610153) or mouse anti-α-Tubulin (1:10000, Sigma-Aldrich, T9026) antibody overnight and with the peroxidase-conjugated donkey anti-mouse antibody (1:5000, Jackson) for 1 hr. The antibodies were diluted in 5% BSA in TBS-T. Bands were visualized using the Western Lightning Plus-ECL substrate (PerkinElmer) for 5 min and a Fusion SL imaging system (Vilber).

iMDDCs were left untreated, or treated with cocaine in indicated concentration. After indicated incubation time, samples were collected in RIPA buffer. Protein lysates were separated on NuPAGE precast gels (Life Technologies Corp.), transferred to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and probed with appropriate primary antibodies followed by incubation with their respective secondary antibodies. Proteins were visualized with Western Lightning Plus ECL Substrate (PerkinElmer, Waltham, MA).
For immunoprecipitation assay, DCs were left untreated or treated with cocaine (1 μM) and incubated for times indicated. DCs were lysed with cell lysis buffer (Cell Signaling Technology). Immunoprecipitation was performed as previously described61 (link).

Cell culture reagents and buffers were purchased from Invitrogen/ThermoFisher (Waltham, MA, USA), PTH_1–34 from R&D Systems (Minneapolis, MN, USA), Biotin-phenol from Iris Biotech GmbH (Marktredwitz, Germany), TrueBlue^TM from Kirkegaard & Perry Lab, Inc. (Gaithersburg, MD, USA). The 30% hydrogen peroxide solution and the 3,3′,5,5′-tetramethylbenzidine (TMB) solution were from Sigma-Aldrich (Oakville, ON, Canada) and the Western Lightning Plus-ECL substrate, from PerkinElmer (Woodbridge, ON, Canada).