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Fig. 7. Dnd1 binds trim36 directly and anchors trim36 to the vegetal cortex. (A) Schematic of trim36 deletion constructs used in the in vitro pull-down assay. (B) Coomassie Blue staining showing purified GST-Dnd1 protein used in the pull-down assay. (C-E) Real-time RT-PCR showing the ratio between RNAs associated with GST-Dnd1 protein and 5% of RNA input. (C) GST-Dnd1 efficiently pulled down trim36, but not GFP or wnt11. (D) Mutation of the URS in trim36 reduced the binding of trim36 by GST-Dnd1. (E) Fusing the 3′-UTR of trim36 to GFP increased the binding of GST-Dnd1 to GFP RNA. (F) RT-PCR showing that knockdown of Dnd1 had no effect on the level of trim36 mRNA in eggs. (G) In situ hybridization showing that trim36 is no longer localized to the vegetal cortex in dnd1-depleted eggs. (H) Immunohistochemistry showing that knockdown of Dnd1 interfered with the accumulation of Trim36 protein in the vegetal cortex of eggs. Inserts at lower magnification show whole eggs. (I) Immunoprecipitation/western blotting showing that the level of Trim36 protein remained unchanged in Dnd1 depleted eggs. Protein lysates from 50 oocytes or eggs were incubated first with an anti-Trim36 antibody to immunoprecipitate Trim36. Supernatants were then incubated with an anti-Dnd1 antibody to pull down Dnd1. (J) Model of Dnd1 function at the vegetal pole. Dnd1 anchors trim36 within the vegetal cortex resulting in localized translation and vegetal accumulation of Trim36. Trim36 promotes vegetal microtubular array formation required to move and thus activate the dorsal determinants. Without Dnd1, trim36 localization is disrupted, Trim36 protein is diluted, microtubular arrays do not form; dorsal determinants remain inactive at the vegetal pole. |
