P smad2

Human or mouse primary muscle progenitor cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and 1 mM EDTA, pH 7.4) containing 1X protease inhibitor (Roche), 1 mM Phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride and 1 mM sodium orthovanadate. Cell lysates were resuspended in 1X Laemmli buffer (Bio-Rad), boiled for 5 minutes and separated on precast 7.5% or 4-15% TGX gels (Biorad). Primary antibodies were diluted in 5% non-fat milk in TBS + 0.1% Tween-20, and nitrocellulose membranes were incubated with antibody mixtures overnight at 4 °C. HRP-conjugated secondary antibodies (Santa Cruz Biotech) were diluted 1:1,000 in 5% non-fat milk in TBS + 0.1% Tween-20 and incubated for 1 hour at room temperature. Blots were developed using Western Lightning ECL reagent (Perkin Elmer), and analyzed with Bio-Rad Gel Doc/Chemi Doc Imaging System and Quantity One software. Antibodies for phospho-ERK1/2, ERK1/2, pSmad1,5,8 and β-Actin, pGSK3β Y216, and Total GSK3β, were purchased from Cell Signaling. pSmad3 was purchased from Epitomics. Smad2/3, and Dll1 antibodies were from Santa Cruz Biotechnology. GapDH and Nicd1 antibodies were from Abcam, pSmad2 and β-Catenin were from Millipore.

Immunoblotting using whole-cell lysate was done with primary antibodies against α-SMA (1:5000; Sigma), E-cadherin (1:5000; BD Biosciences), vimentin (1:1000; Abcam), fibronectin (1:2500; Abcam), collagen I (1:2000; Abcam), Smad3 (1:2500; Cell signaling technology, Danvers, MA, USA), and phosphorylated Smad3 (p-Smad3; 1:2500; Cell signaling technology), Smad2 (1:2500; BD Biosciences), and phosphorylated Smad2 (p-Smad2; 1:2500; Millipore). The sites of the antigen-antibody reaction were detected with horseradish peroxidase conjugated secondary antibodies (P447, diluted 1:10000; Dako, Glostup, Denmark) using an enhanced chemiluminescence (ECL) advanced detection system (GE Healthcare, Little Chalfont, UK). The band densities were quantified by densitometry and compared to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Computer analysis of band pixel intensities was performed by Scion Image (Scion, Frederick, MD, USA).

Cells were homogenized and lysed using RIPA buffer (Amresco) with protease and phosphatase inhibitors (Sigma and Roche, respectively). After centrifugation at 13,000g for 5 min, protein concentrations were measured using the BCA protein assay kit (Pierce) and lysates were separated on a 4–12% Bis–Tris gels (Invitrogen) using MOPS sodium dodecyl sulfate running buffer (Invitrogen). Proteins were transferred with the iBlot system onto nitrocellulose membranes (Novex) and incubated with antibodies p-Smad2 (1:1000, Millipore) and Smad2 (1:1000, Cell Signaling Technology). Signal intensities were detected using ECL Western blotting detection reagents (Amersham Biosciences) and evaluated by ImageJ.