Rho GTPase activity assay
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Cells were grown in log phase in a 10-cm dish, and were starved in 0.5% serum medium or indicated otherwise for 24 h before lysis in a buffer containing 20 mM Tris HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl2, 1% Nonidet P-40, 10% glycerol, and 1 × protease inhibitor mixture. Lysates were clarified, the protein concentrations were normalized, and the GTP-bound Rac1 in the lysates was measured by an effector domain pull-down assay. For the His6-PAK1 PBD pull-down assay, cell lysates were incubated with Ni2+-agarose-immobilized His6-PAK1 PBD domain (~ 1 μg each) purified from E. coli for 30 min. The Ni2+-agarose co-precipitates were washed twice in the wash buffer and analyzed by immunoblotting with anti-Rac1 monoclonal antibody.
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Applications
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NSC 23766 inhibited cell growth and induced apoptosis. NSC 23766 dose-dependently decreased the viability of MDA-MB-468 and MDA-MB-231 cells, with IC50 of ~ 10 μM, but had little effect on the survival of the MCF12A normal mammary epithelial cells. After 24-h exposure to NSC 23766, MDA-MB-231 cells exhibited an increase from 41% to 65% in G1 phase and a concomitant decrease in S and G2-M phases. 100 μM NSC 23766 induced a six-fold increase of apoptotic MDA-MB-468.
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References:
[1]. Gao Y1, Dickerson JB, Guo F, Zheng J, Zheng Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A. 2004 May 18;101(20):7618-23.
[2]. Yoshida T, Zhang Y, Rivera Rosado LA, Chen J, Khan T, Moon SY, Zhang B. Blockade of Rac1 activity induces G1 cell cycle arrest or apoptosis in breast cancer cells through downregulation of cyclin D1, survivin, and X-linked inhibitor of apoptosis protein. Mol Cancer Ther. 2010 Jun;9(6):1657-68.
[3]. Akbar H1, Cancelas J, Williams DA, Zheng J, Zheng Y. Rational design and applications of a Rac GTPase-specific small molecule inhibitor. Methods Enzymol. 2006;406:554-65.
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