Phosphorylation of p38, JNK1/2, and ERK1/2 is analysed by Western blotting. Briefly, TF-1 cells are cultured for 16 h in RPMI 1640 containing 0.1% FBS and subsequently stimulated for various periods of time with medium or OA (30 ng/mL) or SB 203580(1 μM, 5 μM, 10 μM) plus OA. After harvesting, total cell extracts are prepared by resuspending the cells in 500 μL 1× sample buffer (containing 2% SDS, 10% glycerol, 2% β-mercaptoethanol, 60 mM Tris-HCl (pH 6.8) and bromophenol blue) and lysing the cells by passing them through a 23G1 needle (three times). Cell extracts are directly boiled for 10 min and stored at -20°C. Before loading, samples are again boiled for 5 min and cell extracts are resolved by running 1/10th volume on a SDS/12.5%PAGE gel (acryla-mide:bisacrylamide is 173:1) and transferred to cellulosenitrate membrane. Immunoblotting with the antibodies is performed by standard procedures and detection is performed^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Thirty female BALB/c mice with experimentally induced EM are randomly divided into two groups. The first group is designated as the study group (SB 203580 group, n=15), which is injected i.p. with SB 203580 at a dose of 1 μg/mg on a daily basis (this dose is established in a pre-test). The second group is the positive control group (EM group, n=15), which received no medication except i.p. injection of isotonic saline solution (volume equal to that of SB 203580). The sham-operated mice served as the negative controls (sham-operated group, n=15), which received no medication but did have an i.p. injection of isotonic saline solution (volume equal to that of SB 203580). The injection of vehicle or SB 203580 started 3 days before injection of the endometrial fragments (in SB 203580 and EM groups) or the PBS (in sham-operated group) and lasted for 24 days. Then, the mice are sacrificed by cervical dislocation on the day of the last injection and surgical procedures are performed under aseptic conditions.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Lali FV, et al. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem. 2000 Mar 10;275(10):7395-402.

  [2]. Birkenkamp KU, et al. The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway. Br J Pharmacol. 2000 Sep;131(1):99-107.

  [3]. Zhou WD, et al. SB203580, a p38 mitogen-activated protein kinase inhibitor, suppresses the development of endometriosis by down-regulating proinflammatory cytokines and proteolytic factors in a mouse model. Hum Reprod. 2010 Dec;25(12):3110-6.

  [4]. Badger AM, et al. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther. 1996 Dec;279(3):1453-61.

  [5]. Lin Y, et al. Critical role of astrocytic interleukin-17?A in post-stroke survival and neuronal differentiation of neural precursor cells in adult mice. Cell Death Dis. 2016 Jun 23;7(6):e2273.

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