A has been linked to the beads after incubation with the lysate prcontr Sepharose Strip-glutathione-Sepharose beads at 4 for 4 hours. Closing Lich, the beads were washed twice with STE buffer containing 1% corresponds erg Washed complements. IkB Signaling Pathway The effectiveness of the specific Smad2 siRNA shown in the expression of Smad2. 2C. In addition, we used the specific inhibitor SB431542, TGF-Bl skirts yp I receptor kinase, to provide experimental data on the r The TGF yp-I receptor. For this purpose, cells were pretreated with an inhibitor SB431542 for 1 hour, then stimulated TGF or 5 minutes, and the activation of RhoA and RhoB were both using the Rhoteckin RBD GST pull down assay. Figure 2D and 2E document, RhoA and RhoB was activated after stimulation with TGF n the presence of the inhibitor SB431542.
The effectiveness of the inhibitor in blocking the phosphorylation of Smad2 in shown. 2F. These results clearly show that TGF romotes fast RhoA and RhoB activation via a molecular mechanism that is not the kinase activity of T of ALK inhibition the type I receptor and Smad neither specific R This receptor phosphorylation. TGF nduces rapid activation of Src and Vav2 search for molecular targets that can bind TGF rapid activation of Rho, we for the first time on the m Possible involvement of tyrosine kinases in this process. Were first for this purpose the cells First with the previously genistein general tyrosine kinase inhibitor for 1 hour with 5 ng / ml TGF or 5 minutes, treated, followed by measuring the activity of t of RhoA using GST Rhoteckin RBD Pull-down assay. As shown in Fig.
3A, increasing concentrations of genistein significantly inhibited RhoA by TGF mplying early involvement of the tyrosine kinase effectors in this process. To further explore this finding, we examined the levels of phosphorylation of Src non-receptor tyrosine kinase. We focused on Src for several previous reports have documented c Src regulation of TGF As shown in Fig. 3B, TGF reatment induces a rapid activation of Src tyrosine kinase, such as by the increase in the phosphorylation of Residues Ligands Y418, which is required to the catalytic activity of t is completed shops protected. This activation is achieved after 5 minutes and held up to 60 minutes, w While the inhibitory phosphorylation of Src Y529 Residues Walls was only after 15 minutes of TGF timulation clearly indicates that the activation of Src by TGF s strictly regulated.
In a controlled experiment On we followed the phosphorylation of Smad2 by TGF nd found that Smad2 phosphorylation was detected after 5 minutes, became more evident after 15 minutes and held w During at least 60 minutes. Src activation is the phosphorylation and activation of Rho Vav2 GEF, which can in turn be connected to the activation of small Rho GTPases k Brought into connection. Vav2 was functional with the three most important members of the Rho family, Rac1, Cdc42 and RhoA linked. To the R Explore from Vav2 in the activation of RhoA by TGF We used an affinity t Pr zipitationsassay that specifically recognizes activated RhoA activation GEFsin TGF Cells were treated with an siRNA that targets and downregulates expression of Vav2 transfected clearly shown by qRT-PCR analysis. When the cells transfected with the