Fig. S4

Effect of nrz knock down on cytoskeleton organization in the YSL

(A) Immunoblot showing the content of monomeric tubulin (supernatant) and polymerized microtubules (pellet) obtained by subcellular fractionation of zebrafish embryo lysates. (B) Densitometry measurement showing the percentage of monomeric tubulin expressed as a percentage of total tubulin in each fraction. (C) Alpha tubulin staining showing the effect of nrz silencing on microtubule organization in the yolk cell. Control embryos exhibit a dense microtubule network in the yolk cell (white arrowheads); in nrz morphants this network is severely altered. Embryos treated with Nocodazole (6.5 μM) show a similar phenotype. In contrast, incubation of nrz morphants with Taxol (50 μM) restores the yolk cell microtubule network. Scale bar: 50μm. (D) Immunoblot analysis of the actin content in blastomeres and in the yolk cell of control embryos and nrz morphants. Yolk cell proteins were separated from the blastomeres and yolk cortical actin was immunoprecipitated. Lefts panels: whole protein extracts from blastomeres; right panels: immunoprecipitated cortical actin (IP) from yolk homogenates. Alpha tubulin was used for calibration. The intensity of the actin band appeared to be slightly reduced in nrz morphants, suggesting that some proteolytic cleavage may actually have occurred in nrz morphants; however this partial degradation might not account entirely for the dramatic disappearance of cortical F-actin cables in the yolk cell.