Bca method

Cells expressing S1PR1-eGFP or mS1PR1-eGFP were S1P-starved and mock-stimulated or stimulated with the indicated agonists for 5 or 30 min; 10⁶ cells were used for each experimental point. Cells were washed twice in ice-cold phosphate-buffered saline and lysed in 20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 0.15% Nonidet P-40, 1× HALT™ Proteases and Phosphatases inhibitor cocktail, 1.25 U/mL Benzonase. Clarified lysates were incubated for 1 h at +4 °C with GFP-Trap® resin (Chromotek) and extensively washed with lysis buffer. Finally, the immunopurified material was eluted in 4% SDS and analysed by immunoblot. The equal loading was based on the GFP amount reading the samples in the Cary Eclipse Varian spectrofluorometer with 488 nm λ_Ex and 509 nm λ_Em. In some experiments, cells expressing S1PR1 without the GFP tag were used as control and the equal loading was based on total protein concentration measured with the BCA method (G Biosciences).

Mice cytokine/chemokine magnetic bead panel (MCYTOMAG-70K, Merck Millipore, Darmstadt, Germany) was used for the Milliplex ELISA. The concentrations of 3 human mediators (IL-1β, IL-6, and TNF-α) were measured using a multiplex assay instrument (MAGPIX; Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Standard curves of known concentrations of recombinant human cytokines (R&D Systems, Minneapolis, MN) were used to convert fluorescence units to cytokine concentration (picograms/milliliter). The protein concentration of each sample was determined using BCA method (G-Biosciences, St. Louis, MO, USA).