To additional analyze the effects of SB 525334 on kidneys, 9 month previous male Eker rats have been given plain drinking water or the compound in drinking water at 200 mg/L for 2 months. Rats have been then sacrificed and tissues have been harvested, fixed, and stored as described above. For Natural products histology, natural product library tissues have been stained with H&E, and kidneys and multiple sections of female reproductive tract had been examined microscopically by a pathologist blinded as to treatment group. All tumors and proliferative lesions had been identified and evaluated as previously described. In vitro analyses. In vitro experiments had been conducted to examine the effects of SB 525334 on cells from the Eker rat leiomyoma derived cell line, ELT 3. Cells were maintained in DF8 medium for 24 h, then starved in DMEM/F12 medium 1% fetal bovine serum for 24 h.

To determine dose response of ELT 3 cells to SB 525334, cells were treated for 1 h with vehicle, TGF h3, and SB 525334 at 0. 5, 1, and 2 Amol/L, respectively, or TGF h3 SB 525334 at 0. 5, 1, or 2 Amol/L, then harvested for Western analysis for quantitation of SMAD phosphorylation. Treatment with 2 Amol/L of SB 525334 resulted in maximal inhibition Gene expression of phosphorylation and the 2 Amol/L dose was used in subsequent experiments. Western analysis. Purified rabbit IgG antipeptide antibodies to human TGF h1, TGF h2, and TGF h3 were non?cross reacting and have been previously described. Rat leiomyoma and myometrial tissue lysates were subjected to SDS PAGE and transferred to polyvinylidene difluoride membranes.

The membranes were incubated in 3% nonfat dry milk blocking buffer overnight at 4jC and separately incubated with each anti?TGF h isoform antibody in blocking buffer for 3 h, followed by streptavidin horseradish peroxidase?conjugated goat anti rabbit secondary antibody for 1 h Decitabine 1069-66-5 at room temperature, and finally, the Super Signal West Dura Kit was used for detection on X ray film. The protein bands have been quantified by densitometry using an EDAS 290 and the Kodak 1D3. 6 image analysis software. The blots have been stripped and reprobed with an antibody to g tubulin. The net intensity for each band was obtained by comparison with tubulin for each sample and blot. Other antibodies used for Western analysis according to the instructions of the manufacturer have been: TGF h receptor type I and type II, SMAD2/3, and phospho SMAD2. Cell fractionation. To examine phospho SMAD2, SMAD2/3, and TGF h receptor type I and II localization, ELT 3 cells had been treated for 1 h with vehicle, TGF h3, SB 525334, or TGF h3 SB 525334, and harvested for fractionation. For whole cell extracts, cells have been washed twice with ice cold PBS, scraped into 200 AL of cold 1 lysis buffer, homogenized by sonication and pelleted by centrifugation at 14,000 rpm at 4jC for 10 min.