Phospho specific p44 p42 map kinase

Tramadol hydrochloride, naloxone hydrochloride dihydrate, PGF_2α and hydroxyfasudil (fasudil) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Morphine hydrochloride was obtained from Takeda Pharmaceutical Co., Ltd. (Osaka, Japan). SP600125 and SB203580 were purchased from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). The antibodies against phospho-specific SAPK/JNK, SAPK/JNK, phospho-specific p44/p42 MAP kinase, p44/p42 MAP kinase, phospho-specific p38 MAP kinase, and p38 MAP kinase were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). The antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and myosin phosphatase target subunit (MYPT) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Chalfont, UK). Other materials and chemicals were obtained commercially. PGF_2α was dissolved in ethanol. Tramadol, SP600125 and SB203580 were dissolved in dimethyl sulfoxide. Morphine was dissolved in mast cell medium (150 mM NaCl, 5 mM KCl, 5.5 mM glucose, 0.8 mM MgSO₄, 1 mM CaCl₂ and HEPES, pH 7.4). The maximum concentration of ethanol or dimethyl sulfoxide was 0.1%, which has no effects on the assay for OPG or the detection of the protein level using a Western blot analysis (Kuroyanagi et al., 2014 ).