Ine. RAW 264.7 cells were transfected as above, au He f that serum Fetal bovine serum was cultured inactivated at 65 for 1 h before use. Both cell lines were cultured in a humidified atmosphere with 5% CO2 re grown at a constant temperature of 37. HaCaT cells were serum-free for 16 hours before treatment with ligands or inhibitors. Isoliquiritigenin inhibitor Unless otherwise indicated, the cells were treated with low molecular weight inhibitors or appropriate L Solvent treated control 2 h prior to treatment of cells with BMP-2, TGF DAP or meso for 1 h. The cells were then washed once with ice-cold PBS and ice-cold lysis buffer in 0.5 ml of complete full 2-mercaptoethanol, 1 tablet per 25 ml protease inhibitor cocktail. The extracts were min at 16,000 g at 4 for 10 before fracture centrifuged freezing in liquid nitrogen and stored 0 if not treated immediately.
2.5. SDS-PAGE and Western blot of cell extracts were heated at 95 for 5 minutes in 1 x SDS sample buffer glycerol, 2% SDS, 0.02% bromophenol blue and 1% mercaptoethanol, gel St on a polyacrylamide gel Neuroscience to 10% by electrophoresis and nitrocellulose membranes transferred. The membranes were blocked nonfat in TBS-T buffer with 10%. The membranes were then given with the rpern Antique, In TBS-T containing 10% milk for 16 h diluted incubated at 4. The membranes were washed, 2 10 min in TBS-T buffer, probed with secondary Rem Antique Body for 1 h at room temperature, and w Deleted 3 10 min in TBS-T buffer. Detection was performed using verst Rkter chemiluminescence reagent for HRP-conjugated secondary Rantik Body and with the Odyssey Imaging System for 800 or 680-conjugated antibody IRDye Body.
For IC 50 determinations were the intensity Th of the bands, the phosphorylated Smad Smad reasonable and appropriate total land Surface again with the Odyssey imaging system software. 2.6. Tests kinase ALK2, ALK3 and ALK4 ALK5 N-terminal GST constitutively active mutant of ALK3 labeled, wild-type and GST were ALK5 wild-type GST-BMPRII into the pFastBac baculovirus vectors cloned and expressed in Sf9 insect cells. To test kinase, 20 reactionswere the configuration, consisting of 150 ng kinase and 2 g of protein in a buffer containing substrate 50mMTris HCl pH 7.5, 0.1% 2-mercaptoethanol, 0.1 mMEGTA, 10 mM MgCl 2, 0.5 M Microcystein LR, 0.1 mM ATP 32P and 5% DMSO or DMSO containing the appropriate concentrations of low molecular weight inhibitors.
To test Alk2 and ALK3, Smad1 GST was used as substrate. Test ALK4 and ALK5, GST Smad2was used as substrate. ALK3 tests also contained 150 ng of GST BMPRII. The tests were carried out at 30 for 30 min and stopped by adding 1 x SDS sample buffer and heating at 95 for 5 min. The samples were analyzed by SDS-PAGE gels with Coomassie Blue found Rbt and dried gel St. The radioactivitywas analyzed by autoradiography. For IC 50 determinations and the proportion of the remaining Kinaseaktivit t, which found Rbten gangs that protein substrates are removed and the radioactivity t were measured. Third Results 3.1. Specific inhibitors of the TGF-way Although several small molecules were identified as inhibitors as specific inhibitors of the TGF, SB 431 542, SB 505 124, LY 364 947 and A have been reported at 01 83 h Ufigsten used during the tests. In vitro it inhibits ALK5 with an IC 50 of 0.058 M, comparable to the performance of SB 505,124th Fu