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Fig. 1. Antibody specificity. (A) An autoradiogram of SDS-PAGE-separated TnT-synthesized XTCF4, XLEF and XTCF3 polypeptides. (B) A blot of a parallel gel probed with the anti-XTCF3n antibody; the antibody reacts strongly with XTCF3 and more weakly with XTCF4. (C) A blot of a parallel gel probed with the anti-XTCF3c antibody; the antibody reacts with XTCF3 but not XTCF4. Neither anti-XTCF3 antibody reacts with XLEF. (D-F) Embryonic lysates, prepared from unfertilized eggs (egg), two-cell (2C), 32-cell (32c), 64-cell (64c), stage 7 (st7), stage 8 (st8), gastrula/stage 10 (st10) and neurula/stage 13 (s13) embryos were separated by SDS-PAGE, blotted and probed using the anti-TCF3n (D), anti-TCF3c (E) or anti-XSOX3c (F) antibodies. Each lane is loaded with one embryo equivalent. Both anti-XTCF antibodies reacted with a ~67 kDa polypeptide that we presume to be XTCF3 (D,E, arrowhead). The anti-XTCF3n antibody reacted weakly with a 54 kDa polypeptide (D, **) that might be maternal XTCF4 polypeptide. The XTCF3c antibody reacted with distinct sets of lower molecular weight bands (E, *). Anti-XSOX3c (F) reacts strongly with a ~35 kDa polypeptide (arrowhead) throughout early development. The level of this polypeptide decreases in neural stage embryos. In the unfertilized egg (egg) anti-XSOX3c reacts with slower migrating bands (arrows). Occasionally, such slower migrating, anti-XSOX3c-reactive polypeptides are seen in embryonic lysates (arrow). (G) Anti-XTCF3n (Tn) and anti-XTCF3c (Tc) antibodies immunoprecipitated (IP) a common anti-XTCF3n-reactive ~67 kDa polypeptide (arrowhead) present in embryonic lysates (Lys, arrowhead). This polypeptide was not immunoprecipitated by the anti-XSOX3c antibody (Sx). (H) Anti-XSOX3c antibody precipitates a single ~35 kDa, anti-XSOX3c-reactive polypeptide (arrow) that migrates with the 35 kDa polypeptide seen in lysates (Lys, arrowhead). No such band is seen in anti-XTCF3n (TCF3n) or anti-XTCF3c (data not shown) immunoprecipitates. The asterisk (*) marks the heavy and light chains of the precipitating antibody.