Membrane AT1 SB-207499 phosphodiesterase(pde) receptor AT1 receptors were to block the cells pretreated with candesartan. At the end of the experiment, cells were collected media and the cells were used to prepare the whole cell lysates or cytosolic and nuclear fractions or. The samples were stored at until analysis. 2.4. The measurement of the Ang II levels in the ELISA. Ang II levels in the media and cell lysates were measured by competitive inhibition ELISA as previously described by us. Briefly, the samples and standards and to thwart of Ang II and Ang II antibody Body biotinylated peptide in a 96-well plate for 2 h by incubation with horseradish peroxidase for 1 h incubated at streptavidinconjugated followed, room temperature . The final reaction in the well was terminated with 3,3,5,5 tetramethylbenzidine substrate, with 2 N HCl developed and read at 450 nm using an ELISA Leseger t.
Ang II levels in the samples were calculated from a standard curve of Ang II at each test. 2.5. Measurement of matrix proteins And cell proliferation. BI6727 Cell media were dialyzed, lyophilized, and with a known protein concentration. TGF B1 were used by a sandwich that a primary Re-capture Antique Body and avidin-biotin peroxidase detection system measured ELISA. IV collagen and fibronectin in the media cell levels were measured by ELISA using commercially Ltlichen kits from Chemicon and Exocell. To determine cell proliferation were mesangial cells in 96-well plates seeded before the test 24 48 t. The cells were transfected with the Ang II with proteotypic juice and at 37 CO2 emissions by 5% and 95% air for 48 hours, after which cell proliferation was measured with a colorimetric method.
2.6. Course of Study Jak2/Stat3 2.6.1. Protein expression of JAK2 and STAT3. Total cell lysates from mesangial cells treated with Ang II or transfected with exogenous Ang II in RIPA buffer prepared and analyzed for protein expression of JAK2 and Stat3 by Western blotting. The samples were subjected to electrophoresis on acrylamide gel, 8 to 10% and proteins Subjected transferred to a nitrocellulose membrane. Incubation with anti-JAK2 or anti-Stat3 antibody Body was incubated overnight at 4 by washing and incubation with secondary Ren Antique Rpers, followed AHRP. The same membranes were stripped off and determined the expression of proteins for actin. Protein bands were visualized using chemiluminescent substrate and by image analysis.
The results are expressed as the ratio Expressed ratio of JAK2 / STAT3 and actin / actin. 2.6.2. The phosphorylation of STAT3. The phosphorylation of Stat3 was measured using a cell-based assay. Briefly, mesangial cells were seeded in 96 of 24 cell culture plates 48 h before the test t. The cells were divided into two groups and were treated with Ang II or transfected with exogenous Ang II for 20 minutes, after which the medium was removed and cells were incubated with 4% phosphate-buffered formaldehyde/1x a Salzl Solution. After washing and blocking a set of cells for 1 h at room temperature was incubated with phospho serine 727 phospho Stat3 or Stat3 tyrosine 705 Stat3 antibody Measure body phosphorylated, and the other set of cells was treated with a pan Stat3-Antique Body to measure total Stat3. This was accomplished by incubation with HRP-conjugated secondary Rem Antique Body for 1 h at room temperature. The final reaction was