iseases, mediated through MAPK dependent activation of NF ��B pathway in bEnd. 3 cells. Pharmacological approaches suggest that tar geting CO 2 PGE2 system and their upstream signaling components should yield useful therapeutic targets for brain injury and inflammatory diseases. Methods Materials Dulbeccos modified Eagles medium F 12 medium, fetal bovine serum, and TRIzol were from Invitrogen. Hybond C membrane and enhanced chemiluminescence Western blot detection system were from GE Healthcare Biosciences. Anti CO 2 monoclonal anti body was from BD Transduction Laboratories. Phospho ERK1 2, phospho p38, phospho JNK1 2 antibody kits were from Cell Signaling. p65, p42, p38, and JNK1 antibodies were from Santa Cruz. Anti glyceraldehyde 3 phosphate dehydrogenase antibody was from Biogenesis.

BQ 123, BQ 788, GP antagonist 2, GP antagonist 2A, U0126, SB202190, SP600125, and Bay11 7082 were from GSK-3 Biomol. Bicinchoninic acid protein assay reagent was from Pierce. ET 1, enzymes, and other chemicals were from Sigma. Mouse brain microvascular endothelial cell culture Mouse brain microvascular endothelial cells were purchased from Bioresource Collection and Re search Centre and grew in DMEM F 12 containing 10% FBS and antibiotics at 37 C in a humidified 5% CO2 atmos phere. The cell line is acquired from mouse BALB c strain brain cerebral corte endothelial polyoma middle T antigen transformed, which was performed STR PCR profile at BCRC. All the e periments were performed using this cell line and approved by the ethic approval of Chang Gung University. Confluencent cells were released with 0.

05% trypsin 0. 53 mM EDTA for 5 min at 37 C. The cell suspension was plated onto 6 well culture plates or 10 cm culture dishes for the measurement of pro tein or RNA e pression, respectively. Culture medium was changed after 24 h and then every 3 days. E peri ments were performed with cells from passages 5 to 13. Preparation of cell e tracts and Western blot analysis Growth arrested cells were incubated with ET 1 at 37 C for various time intervals. The cells were washed with ice cold phosphate buffered saline, scraped, and collected by centrifugation at 45,000 g for 1 h at 4 C to yield the whole cell e tract, as previously described. Samples were analyzed by Western blot, transferred to nitrocellulose membrane, and then incubated over night using an anti CO 2, phospho ERK1 2, phospho p38 MAPK, phospho JNK1 2, p42, p38, JNK1, p65, or GAPDH antibody.

Membranes were washed with TTBS four times for 5 min each, incubated with a 1 2000 dilu tion of anti rabbit horseradish pero idase antibody for 1 h. The immunoreactive bands were detected by ECL reagents. Total RNA e traction and gene e pression For reverse transcription PCR analysis, total RNA was e tracted from mouse brain endothelial cells stimulated by ET 1, as previously described. The cDNA obtained from 0. 5 ug total RNA was used as a template for PCR amplification. Oligonucleotide primers were designed based on Genbank en