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P. Bargagna–Mohan, Y. Yokota, K. Kim, R. Mohan; Identification of Biological Targets of the Angiogenic Agent Withaferin A . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1756.
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We previously showed that Withaferin A (WA), a small molecule natural compound of the withanolide family, potently inhibits corneal neovascularization. WA exerts its activity by down regulating expression of key stress and inflammatory genes in vascular endothelial cells. To characterize the biological target(s) of WA, we propose to synthesize a cell permeable probe analog of the natural product for use as an affinity reagent for the identification and isolation of the WA–binding protein(s).
We chemically synthesize a biotinylated analog of WA. We used primary endothelial cells from umbilical vein (HUVECs) for our studies. To detect WA’s binding protein(s), confluent plates of HUVECs were serum starved for 24h and then treated with increasing doses of WA–biotin in presence or absence of free WA. Total cell lysates were fractionated on 4.5–20% SDS–polyacrylamide gels followed by protein transfer onto nylon membranes. Biotinylated proteins on the blots were visualized using avidin–horse radish peroxidase and chemiluminescence reagents. Alternatively, the biotinylated proteins were subjected to avidin–sepharose affinity chromatography and purified proteins were analyzed by SDS–PAGE with Coomassie blue staining.
We detected at least two biotinylated protein bands in the avidin–HRP blots that bound specifically to the WA–biotin affinity reagent in a dose dependent manner. Furthermore, WA–biotin affinity chromatography also showed the pull down of specific proteins in a dose–dependent manner. These proteins of interest have been isolated and prepared for characterization by MALDI mass spectroscopic analysis.
The chemical genetic approach to identify novel proteins and study their antiangiogenic function has emerged as a feasible goal with the withanolide class of angiogenesis inhibitors.
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