Mini protean tetra cell blot apparatus

Cells grown on a 15-cm dish were directly scraped off in 200 μl lysis buffer (pH 7.5) containing 150 mM NaCl, 2M urea, 20 mM Tris, 2 mM EDTA, 2 mM EGTA, 3.5% SDS, and 1.5% β-mercaptoethanol, mixed with 6× SDS sample buffer (500 mM Tris–HCl [pH 6.8], 600 mM DDT, 10% SDS, 0.1% bromophenol-blue, and 30% glycerol), DNA sheared by pressing the samples through a 27-gauge needle, and samples were incubated for 5 min at 95°C (Winter et al, 2014 (link)). Serial 5-μm cryosections of frozen skeletal-muscle tissue (quadriceps femoris) were homogenized in lysis buffer, mixed with 6× SDS sample buffer, and incubated for 5 min at 95°C. SDS–PAGE was performed as described (Laemmli, 1970 (link)). Proteins were transferred to nitrocellulose membranes (Protran 0.45 NC; Amersham) using a Mini-PROTEAN Tetra Cell blot apparatus (Bio-Rad).

Cell and tissue lysates were prepared as described in [16 (link)]. Dissected muscles were snap frozen in isopentane cooled with liquid nitrogen, ground in a mortar, and homogenized in lysis buffer (5 mM Tris-HCl pH 6.8, 10% SDS, 0.2 M DTT, 1 mM EDTA, 100 mM NaF, 50 mM ß-glycerophosphate, 2 mM Na₃VO₄, 1 mM PMSF, and cOmplete mini protease inhibitor cocktail (Roche)) using a Dounce tissue grinder. The homogenate was centrifuged for 10 min at 10,000× g, and the supernatant was mixed with 6× SDS sample buffer (500 mM Tris-HCl pH 6.8, 600 mM DDT, 10% SDS, 0.1% bromophenol blue, 30% glycerol). Cells were directly scraped off in 6× SDS sample buffer and DNA was sheared by pressing the samples through a 27-gauge needle. Samples were boiled at 95 °C for 5 min before being subjected to SDS polyacrylamide gel electrophoresis performed under standard conditions. Proteins were transferred to nitrocellulose membranes (Protran 0.2 NC, Amersham Biosciences, Amersham, UK) using a Mini PROTEAN Tetra Cell blot apparatus (Bio-Rad, Hercules, CA, USA). Membranes were scanned, and the amounts of protein contained in individual bands were quantified using ImageJ software (NIH, Bethesda, MD, USA).