Anti myod

To determine protein expression, cells were harvested in RIPA buffer containing a protease inhibitor cocktail, and total protein was quantified using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). Aliquots containing 8 μg total protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then electro-blotted onto a 0.45-μm PV membrane (Immobilon™; Merck Millipore, Darmstadt, Germany). The membranes were blocked and probed overnight with the primary antibodies anti-SEPP1 (1:1000, #ab193193; Abcam, USA), anti-MyoD (1: 1500; Invitrogen, Carlsbad, CA, USA), anti-MyoG (1: 1500; Invitrogen, Carlsbad, CA, USA), anti–β-catenin (1:5000, #ab32572; Abcam, USA), and anti–active β-catenin (1:500, #05-665; Merck Millipore).

According to previously established procedures, cells and muscle samples were harvested, washed with 1× PBS, and lysed in NP40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, and 10% glycerol) with protease inhibitor cocktail (Sigma).⁴¹ (link) Proteins were separated in SDS-PAGE, transferred, and immunoblotted with various antibodies. The antibodies used were anti-MyoD (1:1,000; Invitrogen) and anti-β-actin (dilution 1:3,000; Santa Cruz Biotechnology).