Carcinoma cells were obtained from American ICG-001 Wnt-beta-cetenin inhibitor Type Culture Collection and cultured in DMEM, erg Complements with 10% FBS, glutamine and penicillin / streptomycin at 37th Transient transfection into JEG3 cells with siRNA contr SiRNA or negative, on the Smad2 or Vav2 were carried out in two rounds with Lipofectamine 2000 according to the manufacturer’s instructions. Quantitative reverse transcription-PCR RNA extraction was performed with the kit representation RNAspin mini RNA isolation according to claim manufacturer’s instructions. The first-strand cDNA was synthesized by reverse transcriptase Superscript II and qRT PCR was performed using Maxima SYBR Green / ROX qPCR Master Mix. Housekeeping gene actin was used as contr The mRNA for internal normalization.
Determination of GTP-loaded RhoA, Rac1 and RhoB, Cdc42 levels affinity t F Precipitation with GST beads Rhoteckin RBD proteins, cells were transfected fa Are paid off Accessible, either in two rounds of siRNA contr Negative or siRNA specific targeting Smad2 or Vav2 with Lipofectamine 2000 were serum starved for 24 hours, then stimulated with 5 ng / ml TGF r not for 5 minutes or alternatively pretreated with inhibitors SB431542, PP2 and genistein, then stimulated with 5 ng / ml TGF r not for 5 minutes. The cells were then phosphate buffered saline with ice-cold Solution washed and lysed with cell lysis buffer. Equal volumes of gel Deleted cell lysates were incubated with 20 balls of the GST proteins Rhoteckin RBD on glutathione-Sepharose at 4 for 1 hour.
The beads were washed three times with wash buffer in sample buffer and analyzed in parallel with equal volumes of total cell lysates on a 12% polyacrylamide gel by SDS-PAGE followed by immunoblotting with RhoA or RhoB of the Antik Rpers. The same procedure was used to determine the activity t of Rac1 and Cdc42 with rain pearls t GSTPAK PDB proteins On glutathione-Sepharose and antique Body, specifically followed for Rac1 and Cdc42 in Western blots. Quantification of immunoblots was performed with Tina scan v.2.07d software. Were affinity TSPR Zipitation the active Vav2 GEF with GST-Sepharose beads RhoA affinity TSPR Zipitation the active Vav2 GEF with the mutation, nucleotide free form of RhoA, GST RhoA bound to Sepharose beads, the cells from serum starvation for 24 hours , then with 5 ng / ml TGF or treated at different times.
The cells were then washed with ice-cold phosphate buffered saline Solution and lysed with lysis buffer. Cell lysates were incubated with 20 g offset pearl fra YEARS Riger prepared RhoA GST-Sepharose at 4 for 1 hour. The beads were then washed 3 times with lysis buffer, sample buffer and analyzed in parallel with whole cell lysates on a 10% polyacrylamide gel in SDS-PAGE by immunoblotting with antibodies Rpern and then End Vav2. For the production of GST-Sepharose beads RhoA, competent bacteria with the expression vector transformed pGEXGST RhoA. The bacteria were left in a culture of 200 ml to grow at 37 for about 2 hours until OD600nm0.6 and RhoA protein expression GST induced with 0.5 mM IPTG at room temperature for 16 to 30 hours or 5 hours. The bacteria were then resuspended in 10 ml of STE buffer. The protein was extracted from the bacteria GSTRhoA after sonication and the addition of 1% Triton X-100 by incubation on ice for 20 minutes. The protein