Ultima gold

In vitro methylation of the 72 nt RNA substrate using the Erm protein was carried out using a slightly modified version of a previously described procedure (27 (link)). The reaction was performed in 50 μL volumes containing 50 mM Tris-HCl (pH 7.5), 4 mM MgCl₂, 40 mM KCl, 10 mM dithiothreitol, 3.3 pmol SAM (specific activity, 80 Ci/mmol; PerkinElmer), 10 pmol 72 nt RNA substrate, and 10 pmol purified Erm proteins for 1 h. Reaction mixtures containing everything except proteins were prewarmed to 37°C by at least 5 min of incubation, then the purified Erm proteins were added to prewarmed tubes to minimize any lag in the start of the reaction. After 1 h of incubation, 0.5 mL of ice cold 12% trichloroacetic acid was added in order to terminate the reaction. The methylated RNAs collected by centrifugation were washed twice with 1.25 mL of ice-cold 6% trichloroacetic acid. After drying, the precipitate was extracted with 3 mL of scintillation fluid (Ultima Gold; Packard) and transferred to a counting vial. The remaining precipitate was extracted again with 75 μL of double-distilled water (DDW) warmed to 50°C to 60°C and then extracted once again with 25 μL of prewarmed DDW. All the extracts were pulled together, mixed well, and counted (Tri-Carb 2900TR; Packard, Shelton, CT, USA). Experiments were repeated at least thrice.

Sulfated glycosaminoglycans synthesized by the cells were metabolically labeled with [³⁵S]-sulfate (150 µCi/ml) in F-12 medium for 18 h at 37°C in 2.5% CO₂ atmosphere. Afterwards, the culture medium was removed and the cells washed twice with F-12 medium and scrapped from the dish with 3.5 M urea in 25 mM Tris-HCl pH 7.8. Both to the medium and the cell extract 100 µg of carrier heparan sulfate, dermatan sulfate and chondroitin sulfate were added. The radioactive glycosaminoglycan free chains were prepared from the cell lysates and from the culture supernatants by incubation with 4 mg of maxatase (Biocon do Brasil Ltda, Rio de Janeiro, Brazil) for 4 h at 60°C. The [³⁵S]-sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis, as previously described in [42] (link), [43] (link). The radioactive compounds were located by exposure of the gels (after fixation, drying and staining) to Cyclone storage phosphor screen (Packard Institute – Meriden, USA). For quantification, the radioactive bands were scrapped off the agarose gels, and counted in 5 ml of Ultima Gold (Packard) in a liquid scintillation spectrometer (TRI-CARB 2100TR, Packard). Protein was determined by the Coomassie blue assay. The experiments were performed in duplicates and repeated three times.

⁴⁵Ca²⁺uptake was quantified as described by Ismail et al. [29] . To measure the activity of SACs, myotube cultures were washed twice with PSS containing 1.2 mM Ca²⁺ (PSS+), pre-incubated at 37°C for 15 min with test compounds and then exposed for 5 min to 200 µl/well of a hypo-osmotic PSS+ (100 mOsm obtained by decreasing the NaCl concentration from 145 to 25 mM) containing 1 µCi/ml of ⁴⁵Ca²⁺. Plates were then placed on ice, and cultures were washed 4 times with ice-cold PSS− to remove remaining extracellular ⁴⁵Ca²⁺ before being lysed with 0.5 ml of 1N NaOH. The radioactivity in the lysates was determined by scintillation counting (Ultima Gold, Packard, Groningen, NL) using a beta-counter (LKB Wallac 1217 Rackbeta, Turku, Finland).
To study the activity of SOCs, the cultures were washed twice with PSS+, pre-incubated for 15 min at 37°C with test compounds in 200 µl Ca²⁺-free PSS, and further exposed to 5 µM thapsigargin to deplete intracellular Ca²⁺ stores, in the presence of test compounds. After 10 min, PSS+ containing 1 µCi/ml ⁴⁵Ca²⁺ was added and uptake was measured after another 10 min. ⁴⁵Ca²⁺ was quantified as above.

In vitro methylation of BDV (B. subtilis 23S rRNA domain V, 668 nt) substrates by Erm proteins was carried out by a slightly modified version of a previously described procedure [34 (link),35 (link),48 (link)]. The reaction was performed in 50 μL volumes containing 50 mM Tris-HCl (pH 7.5), 4 mM MgCl₂, 40 mM KCl, 10 mM dithiothreitol, 3.3 pmol S-adenosyl-l-methionine (SAM; specific activity, 80 Ci/mmol; PerkinElmer), 10 pmol rRNA transcripts, and 6.76 pmol (250 ng) purified Erm proteins. Reaction mixtures containing everything except proteins were prewarmed to 37 °C by at least 5 min of incubation, and then purified Erm proteins were added to prewarmed tubes to minimize any lag in the start of the reaction. After 1 h incubation, ice cold 0.5 mL of 12% trichloroacetic acid was added in order to terminate the reaction. The methylated RNAs collected by centrifugation were washed twice with 1.25 mL of ice-cold 6% trichloroacetic acid. After drying, the precipitate was extracted with 3 mL of scintillation fluid (Ultima Gold; Packard), transferred to a counting vial and counted (Tri-Carb 2900TR; Packard, Shelton, CT, USA). Experiments were repeated at least thrice.