Gelatin zymography Brain pericyte conditioned media were concentrated by Amicon Ultra centrifugal filter units, and then put through zymography according to the manufacturers guidelines. Cells were fed every 2 3 times by changing channel. After 10 14 days in culture, floating cells and weakly connected cells of the combined primary cultured cell layer were removed by vigorous shaking of the flask. Then, astrocytes at the bottom of the purchase AG-1478 culture flask were trypsinized and seeded into new culture flasks. The principal cultured astrocytes were maintained in 10 % FBS/DMEM. They were grown in a humidified atmosphere of fifty CO2/95% air at 37 C. Cells in the second or third passage were used for experiments. Western blot analysis Brain pericytes, astrocytes and RBECs were incubated with or without different levels of TNF an at 37 C for the indicated time. When protein kinase inhibitors were applied, they were added 15 min prior to the program of TNF a. To evaluate the expression of TNF a receptor 1 and TNF a receptor 2 among head pericytes, astrocytes and RBECs, these cells were used without TNF cure. The culture supernatants were collected and focused 60 flip applying Amicon Ultra centrifugal filter devices. Cells were scraped and lysed in phosphoprotein lysis buffer Plastid containing 1% phosphatase inhibitor cocktail 1, 1% phosphatase inhibitor cocktail 1% and 2 protease inhibitor cocktail. The total protein concentration in cell lysates was determined utilizing a BCA Protein assay kit. Equal levels of protein from each sample were electrophoretically separated on 5 2006-2007 SDS polyacrylamide ties in, and then transferred to polyvinylidene difluoride membranes. Membranes were blocked with Blocking One or Blocking One G for phosphorylated proteins. Phosphorylation of p42/p44 mitogen-activated protein kinase, p38 MAPK, c Jun N terminal kinase and Akt were found with primary antibodies against phospho p38 MAPK, phospho p42/p44 MAPK, phospho JNK and phospho Akt. MMP 9 and MMP 2 in tradition supernatant were detected using antibodies buy BIX01294 against MMP 9 and MMP 2. TNFR1 and TNFR2 in cell lysates were recognized with an anti MMP 9 antibody and anti MMP 2 antibody. After cleansing, membranes were incubated with the ideal horseradish peroxidase conjugated secondary antibody. To reprobe JNK, p38 MAPK, whole p42/p44 MAPK and Akt, membranes were incubated in stripping buffer for 15 min twice. Akt, p38 MAPK, JNK and whole p42/p44 MAPK were found using main antibodies against p42/p44 MAPK, p38 MAPK, JNK and Akt. The immunoreactive bands were visualized utilizing an ECL Advance Western Blotting Detection Kit. The band pictures were digitally captured with a FluorChem SP imaging process and band intensities were quantified using AlphaEaseFC pc software. The relative strength of phosphorylation of specific proteins was expressed as the ratio of the corresponding total protein and phosphorylated protein.