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Fig. S4. Related to Fig. 4. Confirmation of specificity of Rac, Cdc42, and RhoA antibodies and efficiency of active Rho GTPase GST pull down. (A) Western blots were performed to test the specificity of the αRac1, αCdc42, and αRhoA antibodies. All three antibodies detected endogenous Rac1, Cdc42, and RhoA in un- injected embryos, and levels of these proteins were dramatically increased after injection of mRNA encoding X. tropicalis rac1, cdc42, and rhoa. Arrowheads, the respective protein; −, uninjected embryos; +, injected embryos. (B) To test the specificity and efficiency of the GST pull down, GTPγS was added to protein extracts from embryos injected with 0.5 ng PAK1-GST or rGBD-GST mRNA. Immunoblot was done to detect the respective protein after the GST pull down. The amounts of precipitated (active) Rac, Cdc42, and Rho reveal great increase in GTPγS (+) embryonic lysates compared with untreated lysates. Five percent embryonic lysates were taken as inputs. Arrowhead, the respective protein; −, no treatment with GTPγS; +, treatment with GTPγS; M.W., molecular weight marker. Image published in: Soto X et al. (2013) Copyright © 2013. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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