(C) Western blots show that GQ5 does not affect the expression of Smad4 or Smad7

(C) Western blots show that GQ5 does not affect the expression of Smad4 or Smad7. compound, 3-[(toxicity studies showed that mice intraperitoneally injected with single dose of GQ5 up to 800 mg/kg did not die or develop any obvious adverse event during 7-day observation. GQ5 Ameliorates Renal Interstitial Fibrosis after UUO We initially investigated the effect of GQ5 on renal interstitial fibrosis in UUO, a typical model of renal interstitial fibrosis. In the preliminary study, GQ5 was administered right after operation intraperitoneal injection at the dosage of 10, 20, 40, or 80 mg/kg per day (data indicated that GQ5 selectively inhibited TGF-study, GQ5 did not affect the expression of Smad4 and Smad7 (Figure 4C), nor the phosphorylation of p38, PI3K, or ERK in UUO rats (Figure 4D). Open in a separate window Figure 4. GQ-5 selectively inhibits Smad3 phosphorylation in UUO. Rats receive daily intraperitoneal injection of vehicle or GQ5 (40 mg/kg per day) right after (d1) or 7 days (d7) after UUO and are euthanized 14 days after UUO. (A and B) Immunohistochemical staining (A) and Western blots (B) are performed to analyze the expression of p-Smad3 and p-Smad2 in UUO rats. (C) Western blots show that GQ5 does not affect the expression of Smad4 or Smad7. (D) Western blots show that GQ5 does not affect the phosphorylation of p38, PI3K, or ERK. *and blocking the interaction of Tthat GQ5 could inhibit TGF-receptors, a key event in TGF-receptors with Smads is a complex biologic process involving several adaptor proteins such as SARA,10 embryonic liver fodrin (ELF),36 and kindlin-2.34 In this Grapiprant (CJ-023423) study, we found that GQ5 treatment selectively blocked the interaction of SARA with Smad3, and therefore decreased the binding of Smad3 with Tbinding to its receptor are being developed including decorin,37 soluble chimeric TGF-receptor,38,39 neutralizing antibodies against TGF-(17 kg) (Yunnan Corporation of Materia Medica, Kunming, China) were extracted with 80% ethanol (320 liters) at room temperature. The extracts were concentrated under reduced pressure, and suspended in water followed by partition with ethyl acetate (35 liters). The extract (220 g) from ethyl acetate was submitted to a silica gel column (200?300 mesh, 12150 cm, 2.5 kg; Qingdao Marine Chemical Inc., Qingdao, China), eluted with a gradient of CHCl3/MeOH (100:0?80:20) to yield Grapiprant (CJ-023423) 10 fractions. Fraction 4 (15 g) was subjected to a MCI gel CHP 20P column (75?150 experiments. To test the effect of GQ5 on TGF-for 15 minutes and then heated at 95C and separated on SDS-PAGE gels. Transferred membranes were immunoblotted with the following primary antibodies, respectively: anti-test. Comparisons between groups were made Grapiprant (CJ-023423) using one-way ANOVA followed by the StudentCNewmanCKeuls test. em P /em 0.05 was considered statistically significant. Disclosures We have applied three patents (one international patent and two Chinese patents) for the GQ5 small molecular phenolic compound. The request numbers are PCT/CN2013/087585, 201210111642.X, and 201210111641.5. Supplementary Material Supplemental Data: Click here to view. Acknowledgments This work was supported by grants from the National 973 Program (2012CB517700 and 2011CB504005 to F.F.H.), the Grapiprant (CJ-023423) National Natural Science Foundation of ChinaCJoint Foundation of Yunnan Province (U1202222 to Y.C.), the Nature and Science Foundation of China (81430016 to F.F.H., and 81288001 to J.N.), and the Nature and Science Foundation of Guangdong Province (S2012010009437 to M.L.). Footnotes Published online ahead of print. Publication date available at www.jasn.org. This article Esam contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014040363/-/DCSupplemental..