Anti phopsho jnk

Human Gal-4 was purchased from R&D Systems (Minneapolis, MN, USA). The endotoxin level of Gal-4 was <0.1 EU per 1 μg of protein determined by the Limulus Amebocyte Lysate method. Monocytes were stimulated with Gal-4 for different periods of time and lysed with a lysis buffer from Mammalian Protein Prep Kit (Qiagen, Valencia, CA, USA) in the presence of a protease and phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The monocyte lysates were centrifuged at 12,000g for 15 min. Proteins (20–30 μg) were heated at 100°C for 10 min in SDS sample buffer, separated by SDS-PAGE, and transferred from the gel to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA). Following Abs: anti-ERK, anti-JNK, anti-p38, anti phopsho-ERK, anti phopsho-JNK, anti phopsho-p38,(all from Cell Signaling Technology Inc., Danvers, MA, USA), anti-matrix metalloproteinase (MMP) 2 (Santa Cruz biotechnology, Dallas, TX, USA) and anti-GAPDH (Abcam, Cambridge, MA, USA) were used for detection, and HRP-conjugated secondary Abs purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and Santa Cruz Biotechnology were used for the development of reactions with a chemiluminescent detection system (Pierce Chemical, Dallas, TX, USA).