| |
Fig. S5. Immunodetection of the endogenous Lim1, Otx2, Sia, Mix1 and VegT proteins in X. laevis embryos. (A) Schematic diagrams of antigen regions. A green region of each protein is a part of Lim1 amino acids (aa) 275-403; Accession Number NP_001084128, Sia (aa 1-141; NP_001079305), Mix1 (aa 156−377; NP_001081294), Otx2 (aa 99-288; NP_001084160) and VegT (aa 237-363; NP_001081665), which was used for raising antibodies in rabbits. (B,D,F) Amino acid sequence comparisons. Epitope sequences are indicated by bold and arrows. (C,E,G) Western blotting. Sequence comparison between Lim1 and Lim5/Lhx5 (NP_001084038.1) shows that there is a single stretch of consecutive six amino acids at most in the epitope region (B) and anti-Lim1 antibodies did not crossreact with Lim5 (C). Sequence comparison between Mix1 and Mixer (NP_001081760.1) shows that there is a single stretch of seven identical amino acids at most in the epitope region (D), and anti-Mix1 antibodies did not crossreact with Flag-Mixer. Synthesis of Flag-Mixer was confirmed by western blotting with anti-Flag antibody. Sequence comparison between Otx2 and Otx5 (NP_001081916.1) shows that there are several stretches with more than seven identical amino acids, and anti-Otx2 antibodies crossreacted, though weakly, Flag-Otx5 at the same position as anti-Flag antibody detected. As otx5 is expressed in the organizer in the same manner as otx2, our ChIP-qPCR data for Otx2 included Otx5, but this did not seem to affect the conclusion because Otx5 and Otx2 have supposedly the same function (Kuroda et al., 2000). (H,I) Detection of the endogenous Lim1 and VegT proteins by western blotting. Lysates obtained from 250 gastrula embryos were treated with anti-Lim1 or anti-VegT antibodies, and immunoprecipitates were subjected to western blotting with the same antibodies. One embryo equivalent of lysates from embryos, which had been injected with lim1 or vegt mRNA (100 pg/embryo) was used as positive control. (J-N) Immunostaining for Lim1, Otx2, Sia and Mix1. Bisected gastrula embryos (J-L), the eye at the tailbud stage (M) or tailbud embryos (N) were immunostained with antibodies as indicated. (J′-M′) Higher magnifications of boxed regions indicated in J-M. Staining of animal pole region with anti-Sia antibodies was probably nonspecific, because sia mRNA was not detected by whole-mount in situ hybridization (supplementary material Fig. S6A). The Lim1 protein was detected in the brain (br), spinal cord (sc), notochord (not), prechordal plate (pc) and pronephros (pn) at the tailbud stage (N). (N′,N′′) Higher magnification of boxed regions in N. |
