To determine sellekchem relative gene expression, re sults were analysed using the 2 CT method and normalised to GAPDH expression. Western blot analysis Cells were lysed in 1RIPA buffer containing 1protease inhibitor and 1phos phatase inhibitor, and quantitated using the BCA Protein assay kit. Approximately Inhibitors,Modulators,Libraries 20 to 30 ug of protein was heat denatured at 95 C, separated via SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS Tween for 1 hour and probed with the following primary antibodies at 4 C overnight CTGFCCN2, Smad7, type I collagen, pERK1,2, Erk2, and B tubulin. After washing with TBST, membranes were incubated with the appropriate second ary antibody for 1 hour at room temperature.

Protein levels were visualized by chemiluminscence using the LumiGlo Reserve Substrate and the VisionWorks LS Biospectrum 500 Imaging System. Transient transfections CCD 1068SK fibroblasts were plated at a density Inhibitors,Modulators,Libraries of 2105 cells per well in 6 well plates and allowed to settle over night to reach a final confluence of ca. 50%. For gene knockdown experiments, Transfectin lipid reagent was added in a 2 1 ratio to 20 80 uM CCN2 siRNA or 80 uM Smad7 siRNA, respectively, in serum free DMEM and incubated at room temperature for 20 min before being added drop wise to the cells. Cells were incubated overnight, medium was changed to serum free DMEM and cells were incubated for a further 24 hours before continuing with RNA and protein extrac tions as described above. CCD 1068SK fibroblasts transfected with an equal amount of scrambled control siRNA A were used as a nega tive control.

To transiently overexpress Smad7, 1 ug of the plasmid pORF9 hSmad7 in 150 mM NaCl was added to 2 ul JetPEI reagent Inhibitors,Modulators,Libraries in 150 mM NaCl and incubated at room temperature for 20 min. A total volume of 200 ul transfection mixture was then added drop wise to the cells. 8 h and 48 h post transfection, RNA and Inhibitors,Modulators,Libraries protein were extracted from the cells and used for further analysis as described above. Analysing the incorporation of proline into secreted 1 and 2 procollagen CCD 1068SK fibroblasts at a density of 2105 cells were mixed with an equal number of MCF12A or MDA MB 231 cells, seeded into 6 well plates and allowed to settle overnight. Cells were then washed twice with 1PBS, after which 2 ml serum free DMEM with 20 uCiml proline, 50 mgml ascorbic acid and 50 mgml B aminopropionitrile was added to each well and incubated for 20 hours.

Medium was removed from cells, transferred to 2ml microfuge tube and acetic acid was added to a final con centration of 0. 5 M. Medium proteins were digested Inhibitors,Modulators,Libraries with 100 ugml pepsin for 4 h at 20 C, with rotation. Digested medium was transferred to dialysis tubing and dialyzed overnight against Sorafenib Tosylate price 50 mM Tris, pH 7. 5, with one buffer change after 2 hours. Medium was transferred back into microfuge tubes and precipitated with TCA overnight at 4 C.