Anti p p38 mapk

Total protein of each sample was prepared by standard procedures and quantified by the bicinchoninic acid method (Pierce, Rockford, USA). These protein samples were loaded on 10% polyacrylamide gels and electrophoretically separated. Subsequently, the protein was transferred to polyvinilidene difluoride membranes (Millipore, USA), blocked with 0.1% Tween 20 (TBST) in 5% nonfat dry milk (NFDM) based on Tris-buffered saline (TBS), reacted with primary antibodies (anti-TGF β₁ (1 : 500), anti-BMP-7 (1 : 1000), anti-p38 MAPK (1 : 1500), anti-phosphorylated-p38 MAPK (anti-p-p38 MAPK, 1 : 1000), anti-E-cadherin (1 : 500), anti-α-SMA (1 : 1000), and anti-desmin (1 : 600), Santa Cruz, USA) overnight at 4°C and then HRP-conjugated goat anti-mouse IgG (1 : 3000; Jackson ImmunoResearch Laboratories, Inc., USA) for 2 h at room temperature. After washing, the membrane was processed using Super-Signal West Pico Chemiluminescent Substrate (Pierce), and antibody against GAPDH (Santa Cruz, USA) (1 : 1000) as an internal control [14 (link)].

Colorburst™ electrophoresis marker, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), SB203580 and thrombin were purchased from Sigma-Aldrich/Merck Millipore. Anti p-p38MAPK, horseradish peroxidase-conjugated secondary antibodies and anti-β-actin were purchased from Santa Cruz Biotechnology, USA. The ECL^® system was from GE Healthcare, North Richland Hills, TX, USA. Nitrocellulose membranes (pore size 0.45 µm) were from Bio-Rad Laboratories, Hercules, CA USA.