P jnk1 2

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Escherichia coli lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Interleukin-1 beta (IL-1β), IL-6, IL-10, and tumor necrosis factor alpha (TNF-α) Elisa kits were obtained from eBioscience (Science Center Drive San Diego, CA, USA). COX-2 Elisa kit was purchased from Axygen (Central Avenue Union City, CA, USA). Antibodies against IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, p38α, p-p38α, JNK1/2, p-JNK1/2, ERK1/2, p-ERK1/2, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and secondary antibodies were obtained from Sangon Biotech (Shanghai, China). Other chemicals and reagents were all analytically pure and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin, and penicillin were obtained from Life Technologies (Carlsbad, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IFN-γ, and IL-2 were from R&D Systems (Minneapolis, MN, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from USB Corp. (Cleveland, OH, USA), and the LDH release detection kit was obtained from Roche Applied Science (Indianapolis, IN, USA). All kits were used according to the manufacturers’ protocols. CTX monohydrate, LPS, concanavalin A (ConA), and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). YAC-1 cells, a mouse lymphoma cell line, and RAW264.7 cells, a mouse macrophage cell line, were obtained from ATCC (Manassas, VA, USA; nos. ATCC^®TIB-160^TM and ATCC^®TIB-71 ^TM). Antibodies against β-actin, iNOS, p-NF-κB p65, NF-κB p65, p-IκB, IκB, p-Akt, Akt, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-p38, and p38, and HRP-linked anti-rabbit IgG secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were of the highest grade commercially available.

After being subjected to the indicated treatments, approximate 5 × 10⁶ BM-MSCs were washed with cold PBS three times and lysed with cell lysis solution. Sample buffer was added to cytosolic extracts, and after boiling for 10 min, equal amounts of supernatant from each sample were fractionated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature. Then the membranes were incubated with antibody for p-PERK, PERK, p-IRE1, IRE1, BAX, Bcl-2 (1 : 800; Cell Signaling), caspase-3, eIF 2α, CHOP, p-JNK1/2, JNK1/2, p-extracellular signal-regulated kinase (ERK), ERK, p-p38, p38 (1 : 800; Santa Cruz) at 4°C overnight. At last, immunoreactive bands were then detected by incubation with conjugates of anti-rabbit IgG with horseradish peroxidase (1 : 2,000; Southern-Biotech) for 2 h and visualized by using enhanced chemiluminescence system (ECL; Pierce Company, USA).