Developed with TMB and the absorbance at 450 nm read using an ELISA reader. To each well, the antibody Body reaction was the relative cell number was measured using a normalized Zellf Staining kit.Results as the ratio Ratio of phospho Stat3/total Stat3 are expressed. 2.6.3. DNA-binding activity t of Stat3. Nuclear Cilomilast SB-207499 extracts of mesangial cells were prepared and for determining the activity of t of Stat3 DNA binding. Briefly, samples were incubated nuclear extracts in a 96-well plate with oligonucleotides that are coated, the consensus DNA sequences for binding transcription factor Stat3. Stat3 present in the sample recognized and bound to the consensus sequence-specific DNA and the resulting complex was detected by incubating the samples with a primary DNAStat3 Ren Antique Body Stat3 by incubation with a secondary Ren Antique Body HRP conjugate.
The final reaction was developed with TMB and read at 450 nm in an ELISA Plattenleseger t. Absorbance values represent Bindungsaktivit t of the transcription factor STAT3. 2.7. The statistical analysis. The data were analyzed using student’s ttest and BIRB 796 analysis of variance with post-test comparisons between the groups followed. A pa 0.05 was considered significant. The values are expressed asmean SEM, and n is the number of experiments in each group. Third Results 3.1. Transfection of cells with Ang II HumanMesangial. First, the feasibility of transfection was prime Ren human mesangial cells with Ang II intracellularly Ren Ang II levels are obtained using proteomic juice Hen examined.
Cells in LabTek Kammerobjekttr Were grown like Were mixed with fluorescein-Ang II with proteomic juice for 30 minutes and incubated with labeled examined epifluorescence microscope. 1 shows a sample image of such an experiment. Cells with FITC-Ang II-transfected the presence of green fluorescence was compared to transfected cells. In addition, the cells showed pretreated with 100 M candesartan by transfection with FITC-Ang II, a green fluorescence followed Similar to the observed in transfected cells without treatment, candesartan. These observations suggest that transfection of Ang II to Ang II k proteomic juice Nnte intracellular Re release and Ang II to provide through this process is not affected by treatment with an AT1-receptor antagonists.
To the specific effects of Ang II on intracellular To study re functions of mesangial cells, it was important to the extracellular Re matrix Ang II signaling pathway by binding all the free Ang II in the mixture proteomic juice block at the cell membrane of activated AT1 receptor. For this purpose candesartan to AT 1-receptor-receptor activation due to its physical property, a strong bond prevents weight Hlt and internalisation. Therefore, in all other experiments, mesangial cells were pretreated with candesartan for 1 h and then End transfected with Ang II proteobacterial juice with the transfection reagent. Candesartan had no effect on the proteolytic juice delivery of Ang II on mesangial cells have. 3.2. Delivery of Ang II proteoliposomes Juice obtains ht The intracellular Ren levels of angiotensin II To identify the optimal conditions for the intracellular Re Ang II levels by the transfection procedure proteomic juice obtained To determine hen were mesangial cells with 10 mM glucose alone incubated with NG or proteomic juice and 10 10 M of Ang II for 30 minutes by measuring the intracellular Ren Ang II followed in the cell Ly