Scintillation counter

Proteins from cell lysates (1–2 μg) were incubated for 10 min at 30 °C in 25 μl of a phosphorylation medium containing 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂, 400 μM synthetic peptide-substrate RRRADDSDDDDD and 50 μM [γ-³³P] ATP (1000 cpm/pmol). Assays were stopped by absorption onto phosphocellulose filters. Filters were washed four times in 75 mM phosphoric acid and analyzed by a Scintillation Counter (PerkinElmer).

2 μl of [³H]OA (10 μCi) and 1 μl of 100 mM OA in ethanol were mixed thoroughly in a microcentrifuge tube and then dried under N₂ at room temperature. Then LDs (~10 μg protein) were added to the tube and incubated at 4 °C for 6 h. Then, depending on the experiment, ATP, cytosol or CoA was added to the tube and incubated at 37 °C for various times. After the incubation, LDs were re-isolated by centrifugation at 20,000 g for 3 min and washed with Buffer B 3 times. Total lipids were extracted with acetone-chloroform (2:1, v/v) and analyzed by TLC with a developing solvent of chloroform-acetone-methanol-acetic acid-water (80:40:30:20:10, v/v/v/v/v) for phospholipids, and hexane-diethyl ether-acetic acid (80:20:1, v/v/v) for neutral lipids. TLC plates were stained in iodine vapor after drying at room temperature. Bands corresponding to lipid standard separated on the same plate were scraped, collected and the radioactivity was measured using a Perkin Elmer scintillation counter.

Aliquots (200 μl) of the supernatants from centrifuged lysates that were prepared from incubated muscles were pipetted into a vial with 8 ml of scintillation cocktail (Research Products International, Mount Prospect, IL). A scintillation counter (Perkin Elmer) was used to quantify ³H and ¹⁴C disintegrations per minute. These values were used to calculate [³H]-2-DG uptake according to previously described procedures [30 (link), 31 (link)].

Peptide kinase activity: 2 µg of lysate proteins were incubated for 10 min at 30°C in 25 µl of a phosphorylation medium containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂, 3 µM Staurosporine, 400 µM peptide substrate RRRADDSDDDDD (R₃AD₂SD₅) and 20 µM [γ-³³P]ATP (1000 cpm pmol⁻¹). Assays were stopped by absorption onto phosphocellulose p81 filters (PerkinElmer, Waltham, MA, USA), which were washed four times in 75 mM phosphoric acid and analysed by a Scintillation Counter (PerkinElmer).
Protein kinase activity: 2 µg of lysate proteins were incubated for 10 min at 30°C in 25 μl of a phosphorylation medium containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂, 3 µM Staurosporine, 1 µg β-casein (Sigma-Aldrich) and 20 µM [γ-³³P]ATP (1000 cpm pmol⁻¹). Assays were stopped by adding Laemmli buffer and samples were subjected to 13% SDS-PAGE. Radioactive ³³P- β-casein was evidenced by analysing the dried gel with a Cyclone Plus Storage PhosphorSystem (PerkinElmer).

Primary production was measured twice daily, at the beginning and at the end of the light period, using a slight modification of the ¹⁴C radiolabel method (50 (link)). The total activity of 1.85 kBq H¹⁴CO₃ was added to 1-ml samples. For each incubation bottle, three sets of incubations were prepared for measuring (i) total carbon fixation, (ii) the fraction of primary production released as DOC, and (iii) dark CO₂ assimilation. The samples were incubated next to the experimental units for 2 h in technical duplicates. After the incubations, the samples for the fraction of primary production released as DOC were gently filtered through a 0.2-μm polycarbonate filter into clean scintillation vials, and 100 μl of 1 mol liter⁻¹ HCl was added to all vials to volatilize nonincorporated H¹⁴CO₃. The vials were left for 24 h in an exhaust hood before 4 ml of scintillation liquid (Perkin-Elmer, Waltham, MA) was added. The activity was determined in a scintillation counter (Perkin-Elmer). The total dissolved inorganic carbon concentration (DIC) was calculated based on temperature, pH (Inolab pH 720; WTW Xylem, Inc., Rye Brook, NY), and alkalinity measurements (Metrohm 877; Herisau, Switzerland), and the total carbon fixation rate was calculated knowing the radiolabeled C uptake and the fraction of H¹⁴CO₃ added to the total DIC pool.