Elisa kits for tnf α and il 1β

PQ was purchased from Sigma-Aldrich (St. Louis, MO, USA). CB2R-agonist JWH133 was obtained from Enzo Life Sciences Ltd. (UK). Antibodies to p38MAPK, phosphor-p38MAPK, JNK1/2, phosphor-JNK1/2, ERK1/2, phosphor-ERK1/2, NF-κBp65, IκB-α, phosphor-IκBα, and β-actin were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Rabbit anti-CB2 receptor polyclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The rat MPO determination kit was purchased from Jiancheng Bioengineering Institute of Nanjing (Nanjing, China). The ELISA kits for TNF-α and IL-1β were purchased from R&D system (R&D, USA).

The RASFs were plated in 6-well dishes and grown to confluence. The culture medium was then exchanged with serum-free RPMI medium. Cells were pretreated for 30 min with pharmacological activators Angiotensin II (a FAK activator), 740-YP (a PI3K activator), SC-79 (an AKT activator) and prostratin (an NF-κB activator) to stimulate the RSAFs, before 24 h of Antcin K (0, 0.3, 1, 3, or 10 μM) treatment. The conditioned medium was collected and stored at –20°C until use. Secreted expression of TNF-α, IL-1β and IL-8 was examined using ELISA kits for TNF-α, IL-1β and IL-8 (R&D Systems, MN, USA), according to the manufacturer’s protocols.
Serum levels of TNF-α, IL-1β, IL-8 and IL-6 in CIA mice were examined using ELISA kits for TNF-α and IL-1β (R&D Systems, MN, USA), IL-8 (MyBioSource, Inc, San Diego, USA) and IL-6 (Max™ Set Deluxe Kits, BioLegend, USA) according to the manufacturers’ protocols.

The ELISA kits for TNF-α and IL-1β were purchased from R&D Systems (Minneapolis, MN, USA) and iNOS and NF-κB were obtained from MSK Biological Technology (Wuhan, china), according to the protocol provided by the manufacturer under prescribed 450 nm conditions in an ELISA reader (Bio-Rad, Hercules, CA, USA).