3h ouabain

Analysis of skeletal muscle [³H]ouabain binding site content was performed as described previously by Nørgaard et al. (1984 (link)) to determine muscle NKA content. In brief, 20 mg of muscle was used; each sample was washed for 2 × 10 min at 37°C in vanadate buffer (250 m·molL⁻¹ sucrose, 10 m·molL⁻¹ Tris·HCl, 3 m·molL⁻¹ MgSO₄, 1 m·molL⁻¹ NaVO₄; pH 7.3). Muscle samples were then incubated for 2 h at 37°C in vanadate buffer with the addition of [³H]ouabain (2.0 Ci.mL⁻¹ and 10⁻⁶ molL⁻¹, PerkinElmer, Boston, MA). The muscle was then placed in ice-cold vanadate solution for 4 × 30 min to remove any unbound [³H]ouabain. Muscle samples were blotted on filter paper and weighed before being soaked in 500 μL of 5% trichloroacetic acid and 0.1 m·molL⁻¹ ouabain for approximately 20 h. Following this, 2.5 mL of scintillation cocktail (Opti-Fluor; Packard, PerkinElmer) was added before liquid scintillation counting of [³H]ouabain. The [³H]ouabain binding site content was calculated on the basis of the sample wet weight and specific activity of the incubation buffer and samples and expressed as pmol·g·ww⁻¹. The final [³H]ouabain binding site content was then calculated as described previously (Nørgaard et al. 1984 (link); Petersen et al. 2005 (link)).

Acrylamide and bis-Acrylamide were from SERVA (Heidelberg, Germany). Immobiline DryStrips, Pharmalyte buffer (broad pH range 3–10) and Immobiline DryStrip cover fluid were purchased from GE Healthcare (Piscataway Township, NJ). Complete protease inhibitor cocktail was from Roche Diagnostic (Mannheim, Germany), [³H]-ouabain (30 mCi/mmol, NET211001MC) from Perkin Elmer and N-glycosidase F (11 365 169 001, Roche) was from Sigma-Aldrich. All others chemicals were of highest purity available and purchased from Sigma-Aldrich (St. Louis, USA).