Figure S2. XFDL156 neither Reduces Phosphorylated Smad2 nor Affects Its Nuclear Import (A) Activin-treated animal caps were analyzed by q-PCR with indicated primers. 400 pg/cell of control mRNA (lanes 1 and 2), 100 pg/cell of XFDL mRNA (lane 3), 400 pg/cell of XFDL mRNA (lane 4), 50 ng/cell of p53-MO (lane 5) or 100 pg/cell of Fast1-EnR mRNA were injected into each animal blastomere at the 8-cell stage. Analysis was performed as in Figure 2A. (B) Siamois expression by Wnt8 was not affected by XFDL. 40 pg/cell of Wnt8 (lanes 2 and 3) and 400 pg/cell of XFDL (lane 3) were injected in each animal blastomere at the 8-cells stage and analyzed as in Figure 2A. (C-E) XFDL neither reduced phosphorylated Smad2 nor affected its nuclear transport. (C) Animal caps were prepared from embryos received injections of control (400 pg/cell; lanes 1-4) or XFDL (400 pg/cell; lanes 5-8) mRNA and incubated for 30 minutes with 5 ng/ml (lanes 2 and 6), 20 ng/ml (lanes 3 and 7) or 50 ng/ml (lanes 4 and 8) of Activin. Western blots were performed using anti-phosphorylated Smad2 (top) or anti-Smad2 (bottom) antibodies. (D) Nuclear accumulation of phospho-Smad2 was measured by Western blot. Control embryos (lanes 1 and 2), embryos injected with FLAG-tagged XFDL mRNA (FL-XFDL; 400 pg/cell; lanes 3 and 4) or XFDL-MO (50 ng/cell; lanes 5 and 6) were subject to analysis as described in Experimental Procedures. Arrows, pSmad2 in the nuclear extracts (the lower band presumably represents the alternative splice form devoid of exon 3; Batut et al., 2007); asterisk, non-specific bands. (E) Selective extraction of the nuclear and cytoplasmic protein fractions was confirmed by Western blot with the anti-HSP70 (cytoplasmic; upper) and anti-Histone H3 (nuclear; lower) antibodies. c; cytoplasmic, n;nuclear.
Image published in: Sasai N et al. (2008)
Copyright © 2008. Image reproduced with permission of the Publisher, Elsevier B. V.
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