Pageruler plus

Two-dimensional electrophoresis (2D-PAGE) was performed as described [42 (link)]. Briefly, the first dimension of 2D-PAGE was performed by applying 100 µg of acetone-precipitated protein per sample to pI, 4–7, 7.0 cm immobilized pH gradient (IPG) strips (Immobiline™ Dry Strip, GE Healthcare Bio-sciences AB; Uppsala, Sweden). The strips were rehydrated overnight at room temperature with 135 µL DeStreak Rehydration Solution. Isoelectric focusing (IEF) was performed by using the Ettan™ IPGphor3™ Unit (GE Healthcare Europe; Freiburg, Germany) and carried out at 20 °C for 6.5 h at 5000 V and 50 µA/strip.
Then the strips were sequentially equilibrated for 20 min in 2 mL equilibration buffer 1 (0.05 M trichloroethylene HCl (pH 8.8), 6 M urea, 30% glycerol, 4% SDS, 2% DTE, 0.002% bromophenol blue) and equilibration buffer 2 (0.05 M trichloroethylene HCl (pH 8.8), 6 M urea, 30% glycerol, 4% SDS, 2.5% iodoacetamid, 0.002% bromophenol blue). Standard molecular weight prestained protein ladder marker (10–250 kDa; Page Ruler™ Plus, ThermoScientific; Germany) and IPG strips were loaded onto homogeneous 12% polyacrylamide gels and sealed with 1% agarose solution. Electrophoresis was carried out at room temperature and 10 mA/gel until the tracking dye reached the bottom of the gels (1.5 h). 2D-PAGE protein profiles were visualized using Coomassie blue stain as previously described [43 (link)].

The active forms of simvastatin (hydrophobic) and rosuvastatin (hydrophilic) were purchased from Calbiochem and LKT Laboratories. Both statins were dissolved in DMSO (Sigma Aldrich, St. Louis, MO, USA) and used at 5 µM concentrations after initial testing as recently published [26 (link)]. A skeletal-muscle growth medium was obtained from ProVitro (Berlin, Germany). Opti-MEM and GlutaMax were purchased from Invitrogen (Berlin, Germany). All antibodies are listed in Table 2. The pre-stained protein marker PageRuler™ Plus was from Thermo Scientific (St. Louis, MO, USA), and HiMark™ was obtained from Invitrogen (Berlin, Germany). Krebs Ringer buffer solution at pH 7.4 was composed of 25 mM HEPES, 135 mM NaCl, 3.6 mM KCl, 1 mM CaCl₂, 0.5 mM MgSO₄, 0.5 mM KH₂PO₄, and 5 mM NaOH. Protein lysis buffer at pH 7.4 contained 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, 1 mM PMSF, 1 mM glycerol phosphate, 1 mM Na₃VO₄, 1 mM NaF, 2.5 mM Na₄O₇P₂, 1x PhosSTOP (Roche, Mannheim, Germany), and 1x Complete EDTA-free (Roche, Mannheim, Germany).

White chicken feathers were supplied by a local poultry slaughterhouse (Drobful, Poland). NaOH (POCH, Poland) was used to carry out the hydrolysis. DTT and Coomassie brilliant blue R-250 (Fluka, USA), SDS (Merck, Germany), 30% solution of acyrlamide/bis-acrylamide, ammonium persulfate, bromophenol solution, glycine, hydrochloric acid, TEMED, and trizma base (Sigma-Aldrich, USA), acetic acid, glycerol and methanol (Chempur, Poland), ammonium perfulfate, and prestained protein ladder PageRuler Plus (Thermo scientific, USA) were applied for determination of protein molecular weight. Sulfuric acid and Tashiro’s indicator (POCH, Poland), selenium catalyst (Chempur, Poland), boric acid, and hydrochloric acid (Stanlab, Poland) were used to determine the protein content by the Kjeldahl method.