Ecoscint a

The relative overall transcription rate was compared by determining the rate of ³H-uridine incorporation into the exponential cultures of M. maripaludis grown at 22°C. Portions of exponentially growing cultures at OD₆₀₀ of ∼0.45, 600 μl, were collected at 0, 0.2-, 0.5-, 1-, 1.5- and 2-min post-addition of [5,6–³H]-Uridine (PerkinElmer) at a final concentration of 105 μCi/ml. Total RNA was extracted using TRIzol^TM reagent (Invitrogen) and dissolved in 700 μl Ecoscint A (National Diagnostics). ³H isotope incorporation was determined by liquid scintillation counting (Perkin Elmer). Relative overall transcription rate was calculated from the linear increase in cpm incorporation with time.

For uptake studies, IEC-18 cells were grown on Transwell inserts (insert size 24 mm, pore size 0.4 µm; Corning, Cat# 29442, NY, USA) in 24-well plates. IEC-18 cells were plated with equal numbers of cells. Uptake studies for Na-K-ATPase were done using radioactive Rubidium (⁸⁶Rb⁺, PerkinElmer, Waltham, MA, USA). ⁸⁶Rb⁺ is comparable to K⁺ in chemical characteristics and has similar affinity for the Na-K-ATPase and, hence, was used to determine Na-K-ATPase activity [28 (link),29 ]. Cells were incubated for 1 h in serum-free DMEM (SFM). The cells were subsequently washed with SFM and incubated for 10 min at 37 °C in SFM containing 20 μM monensin on both sides of the membrane. Then, cells were washed with SFM. Na-K-ATPase uptake studies were then performed by incubating cells for 15 min with reaction mixture (SFM and ⁸⁶Rb⁺ (~1 μCi/well)) on the basolateral side of the membrane in the presence and absence of ouabain (1 mM). The reaction was stopped by the addition of ice-cold MgCl₂ (10 mM), subsequently washed three times with MgCl₂, and the cells were lysed with 800 μL of 1 N NaOH and incubation for 30 min at 70 °C, which was then mixed with 5 mL of Ecoscint A (National diagnostics). The vials were kept in darkness overnight, and the radioactivity retained by the cells was determined in a Beckman Coulter 6500 scintillation counter.

To prepare membrane fractions, cells were harvested in phosphate buffer saline (PBS), frozen at least overnight at −80 °C, and then homogenized in ice-cold 50 mM Tris-HCl buffer, pH 7.5 using a Potter Elvehjem tissue grinder. The nuclear pellet was removed by centrifugation at 1000× g for 15 min at 4 °C. The total membrane fraction was collected after centrifugation of the supernatant at 100,000× g for 35 min at 4 °C. The membrane fraction was aliquoted and stored at −80 °C in 50 mM Tris-HCl, pH 7.4, and the protein concentration was determined by the Bradford method. The [³⁵S] GTPγS binding assays were performed in polypropylene tubes in a buffer consisting of 20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 0.1% fatty acid-free BSA, and 10⁻⁴ M GDP. Membranes were incubated for 60 min at 30°C in the buffer supplemented with 5 µg saponin, 0.2 nM [³⁵S] GTPγS, and 1 mM of the S1PRs agonist FTY720-P. The reaction was stopped by vacuum filtration through Whatman GF/B glass filters preincubated in buffer, which were then washed three times with 4 mL of ice-cold buffer without GDP. Membrane-bound radioactivity was determined by liquid scintillation counting (Packard, GMI Trusted laboratory Solutions, Ramsey, MN, USA) after overnight extraction of the filters in 4 mL of scintillation cocktail (Ecoscint A, National Diagnostics, Fisher Scientific).