Muscles were lysed, the supernatant was neutralized, and radioactivity was quantified using liquid scintillation counting.
After incubation with CsA, rabbit papillary muscles were lysed in 50 µl calcineurin assay buffer (BioMol, Plymouth Meeting, PA).
C2C12 cells or regenerating muscles were lysed in Trizol (Invitrogen, Carlsbad, CA, USA), and RNA was isolated following the manufacturer's protocol.
Isolated soleus muscles were lysed in lysis buffer (in mM: Tris-HCl 10, sucrose 250, EDTA 1, EGTA 1, Triton X-100 2%, pH 7.4; and 1 tablet protease inhibitor per 10 mL).
Muscles were lysed in modified RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2% sodium dodecyl sulfate (SDS), 2% deoxycholate, 1 mM PMSF (phenylmethanesulfonylfluoride), 1 mM EDTA, 5 mM sodium fluoride, 2 mM sodium orthovanadate, and protease inhibitors including 1 mM phenylmethylsulfonyl fluoride, 1 μg/μl pepstatin, 1 μg/μl leupeptin, and 2 μg/ml aprotinin).
To determine protein expression, flash-frozen quadriceps muscles were lysed with ice-cold lysis buffer (Nonidet P-40 1%, glycerol 10%, NaCl 137 mM, Tris HCl pH 7.5 20 mM) containing protease (Complete Mini, EDTA-free, Roche) and phosphatase inhibitors (PhosSTOP, Roche).
Briefly, the leg muscle was lysed in RIPA buffer supplemented with protease and phosphatase inhibitor mixture (Sigma, USA), and protein concentrations of cell lysates were determined by BCA kit (Beyotime, Haimen, China).
Muscle tissues were lysed in a buffer containing 50 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 0.5% Nonidet P-40, and protease and phosphatase inhibitors.
Briefly, tissue or L6 muscle cells were lysed in 100 mM Tris HCl (pH 7.4) containing 250 mM sucrose and 2 mM EDTA.
Primary skeletal muscle myotubes were lysed in cap lysis buffer (140 mM KCl, 10mM Tris pH 7,5, 1mM EDTA, 4mM MgCl2, 1mM DTT, 1% NP-40, 1 mM sodium orthovanadate, 50 mM β-glycerophosphate, 10 mM NaF and proteases inhibitors).
Briefly, mice were killed 48 h after the D5-F injection, TCA insoluble total muscle proteins were lysed in 6 M HCl, and total amino acids were purified by passing over cation exchange column (AG50 W-X8, Bio-Rad).
