Gelatin zymography Brain pericyte conditioned media were subjected to zymography according to the manufacturers tips concentrated by Amicon Ultra centrifugal filter units, and then. Cells were fed every 2 3 times by changing method. 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 Imatinib clinical trial culture flask were trypsinized and seeded into new culture flasks. The principal cultured astrocytes were preserved in one hundred thousand FBS/DMEM. They were developed in a humidified atmosphere of fifty CO2/95% air at 37 C. Cells at the second or third passage were used for experiments. Western blot analysis Brain pericytes, astrocytes and RBECs were incubated with or without different concentrations of TNF an at 37 C for the indicated time. When protein kinase inhibitors were applied, they were added 15 min ahead of the application of TNF a. To examine the expression of TNF a receptor 1 and TNF a receptor 2 among brain pericytes, astrocytes and RBECs, these cells were employed without TNF cure. The culture supernatants were collected and focused 60 collapse using Amicon Ultra centrifugal filter devices. Cells were scraped and lysed in phosphoprotein lysis buffer Messenger RNA 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 using a BCA Protein assay kit. Equivalent levels of protein from each sample were electrophoretically separated on 5 two decades SDS polyacrylamide gels, 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 main antibodies against phospho p38 MAPK, phospho p42/p44 MAPK, phospho JNK and phospho Akt. MMP 9 and MMP 2 in culture supernatant were detected using antibodies Icotinib against MMP 2 and MMP 9. TNFR1 and TNFR2 in cell lysates were detected with an anti MMP 9 antibody and anti MMP 2 antibody. After washing, membranes were incubated with the appropriate horseradish peroxidase conjugated secondary antibody. To reprobe total p42/p44 MAPK, p38 MAPK, JNK and Akt, membranes were incubated in stripping buffer for 15 min twice. JNK, p38 MAPK, total p42/p44 MAPK and Akt were found using major antibodies against p42/p44 MAPK, p38 MAPK, JNK and Akt. The bands were visualized using an ECL Advance Western Blotting Detection Kit. The band pictures were digitally taken 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.