Fig. 10. Manipulating maternal XSOX3 activity. (A) Stage 8 embryo lysates (300 µl) were incubated alone (Lys) or together with 0.5 µg anti-XSOX3c (Lys + Sx3') or anti-XTCF3n (Lys + Tf3') antibodies. Lysates were then incubated with either streptavidinagarose beads (SA alone) or biotinylated DC5-streptavidin-agarose beads (DC5-SA). Both lysates and DC5-bound proteins were analysed by immunoblot with anti-XSOX3c. XSOX3 (arrowhead) was bound to DC5 in control and anti-XTCF3n-containing lysates, but its binding was greatly reduced by the addition of the anti-XSOX3c antibody; no binding was seen in the absence of DC5 DNA. (B) Fertilized eggs were injected with anti-XSOX3c antibody (10 nl of a 7.5 mg ml^–1 solution) or anti-XTCF3n antibody (7.8 mg ml^–1 solution). At stage 9, the embryos were homogenized and analysed by DNA fishing with DC5-streptavidin-agarose. Anti-XSOX3c-injected (Sx3), anti-XTCF3n-injected (Tf3) and uninjected (Un) lysates were analysed by immunoblot using anti-XSOX3c. The total amount of XSOX3 was unchanged upon antibody injection, but anti-XSOX3c dramatically inhibited the binding of XSOX3 to DC5-streptavidin-agarose (DC5 fishing). (C) Fertilized eggs injected with either anti-XSOX3c antibody (10 nl of a 7.5 mg ml^–1 solution) (antiSX3), anti-XTCF3n antibody (10 nl of a 7.8 mg ml^–1 solution) (anti-TCF3), XSOX3 RNA (1 ng) (Sx3 RNA), XSOX3C-VP16 RNA (1 ng) (Sx3CVP16) or XSOX3C-EnR RNA (1 ng) (Sx3-EnR) were allowed to develop to stage 9 and then homogenized and analysed by quantitative RT-PCR. XSOX3 and XSOX3C-EnR RNA injection reduced Xnr5 RNA levels; injection of either anti-XSOX3c and XSOX3C-VP16 RNA increased Xnr5 RNA levels; injection of anti-XTCF3n did not alter Xnr5 RNA accumulation.

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