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Fig. S1. Generation of Nkx2.5 Morpholino (MO) and its specificity.
(a) Nkx2.5 MO was synthesized according to a previous study (Nagao et al., 2008). Western blot analysis was performed to examine the effect of Nkx2.5 MO on the translation of Nkx2.5 in oocytes. nkx2.5-myc mRNA (9.2 ng) alone or nkx2.5-myc mRNA together with Control MO (9.2 pmol) or with Nkx2.5 MO (9.2 pmol) was injected into defolliculated Xenopus oocytes. Intact oocytes served as controls. Oocytes were then cultured for 24 hours and were homogenized by pipetting in a lysis buffer (100 mM KCl, 2 mM MgCl2, 1 mM PMSF, 10 mM HEPES-KOH, pH 7.4) (4 μl/oocyte). The soluble protein fraction (20 g/lane) was electrophoresed in 12.5% SDS-polyacrylamide gel under a denatured condition. After blotting the proteins onto an ECL membrane, anti-myc (QE10) monoclonal antibody and peroxidase-conjugated goat anti-mouse IgG were sequentially reacted. After washing, the membrane was reacted with ECL Western Blotting Reagent Substrate for Peroxidase (Amersham) and the signal was detected with Light Capture 2 (Ato). (b) A restoration experiment was performed to show the specific effect of Nkx2.5 MO on nkx2.5 mRNA. For this purpose, Nkx2.5 MO (18.4 pmol/embryo) was injected into the DMZ at the 4-cell stage with (3) or without (2) myc-nkx2.5 mRNA. Intact embryos served as controls (1). These embryos were then fixed at st. 33 and processed for in situ hybridization to analyze the expression of MHC. Expression of MHCwas clearly suppressed by Nkx2.5 MO injection (2), and its expression was rescued by coinjection of myc-nkx2.5 mRNA (100 pg) (3). Percentage of embryos with positive expression of MHCis shown in a bar graph.
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