Información de entrada al modelo de simulación

The antibodies of the Lef/Tcf protein family members were directed against the most divergent domains between the β-catenin binding domain and

the HMG box (see Figure 13). Nevertheless, these regions still share a sequence similarity of about 40%. Therefore, the antibodies were tested for cross-reactivity. In Figure 16, the antibodies LEF 3D4, LEF 5F12, Tcf1 1D12, Tcf1 2F4, Tcf3 3E5 were analyzed by IP with in vitro translated, 35_{S-labeled Lef1, Tcf1, Tcf3 and}

Tcf4a alone or as a mixture. LEF 3D4 and LEF 5F12 were specific for Lef1, Tcf1 1D12 and Tcf1 2F4 for Tcf1. The αLef antibodies enriched the smaller band

of the in vitro translated Lef1 double band. The αTcf3 antibody Tcf3 3E5 did not

Figure 16: Lef/Tcf antibodies precipitate their specific in vitro translated antigen, but no other members of the protein family

2µl of in vitro translated, 35S labeled Lef1 (encoded by plasmid pLef1-FLAG), Tcf1 (encoded by plasmid pTcf1-FLAG), Tcf3 (encoded by plasmid pTcf3-FLAG), Tcf4a (encoded by plasmid pTcf4a-FLAG) and mock control (no DNA) were supplied individually or as a mix of all 5 TNT reactions IPs with the indicated antibodies; Protein-G sepharose beads alone served as a control (beads). In addition to the 50% input, on each gel 10% of the input of the mixed in vitro translation reactions was loaded in the first lane of all gels to make it easier to discriminate between the four Lef/Tcfs. The figure shows an autoradiograph of the 35S-labeling with an exposure time of 24h.

The antibodies LEF 3D4, LEF 5F12, and Tcf1 2F4 precipitated also in vivo specifically their over-expressed antigen out from a mixture of all four Lef/Tcf protein family members (Figure 17). Tcf1 1D12 also precipitated its specific

antigen Tcf1, but less efficiently than Tcf1 2F4. Therefore, the subsequent experiments were performed with Tcf1 2F4 solely. Tcf3 3E5 was again not able to precipitate Tcf3 or any of the other Lef/Tcfs.

Figure 17: Lef1 and Tcf1 antibodies precipitate specifically their overexpressed antigen in vivo

250pg each of in vitro transcribed MT-Tcf1-FLAG mRNA, MT-Lef1-FLAG mRNA, MT- Tcf3-FLAG mRNA and MT-Tcf4a-FLAG mRNA were injected into 4 cell stage embryos and cultured until the blastula stage (NF9). Four eeq Tcf-Mix injected embryo lysate (X) or untreated embryos (UI) were used for the IPs with the indicated antibodies. ProteinG- sepharose beads (Protein G) served as a background control. 33% of the inputs were also loaded on SDS-PAGE. The immunoblotting was performed with αMyc antibody

9E10. IgG-HC: IgG heavy chain.

In addition to the IP specificity in non-crosslinked embryo lysates, also the IP specificities of the antibodies under formaldehyde-fixed ChIP conditions were investigated. Lysates of formaldehyde-fixed, Tcf-mix injected or uninjected embryos were applied to the ChIP-type IP. The αLef1 antibodies LEF 3D4 and

LEF 5F12 precipitated predominantly Lef1. Unanticipated, they also precipitated the other three Tcf proteins Tcf1, Tcf3 and Tcf4a under ChIP conditions

(Figure 18). The αTcf1 antibody Tcf1 2F4 enriched primarily Tcf1 but also the

other three Tcf family members Lef1, Tcf3 and Tcf4a. The αTcf3 antibody

Tcf3 3E5 was able precipitated all 4 Lef/Tcfs, but did not preferentially bind to Tcf3. In order to exclude a DNA-dependent co-precipitation of the Lef/Tcfs, the IPs were performed with and without the presence of DNaseI. This revealed, that the co-precipitation of the Lef/Tcf protein occurred in a DNaseI-treatment independent manner.

Figure 18: Lef/Tcf antibodies precipitate all four over-expressed Lef/Tcf family members upon formaldehyde fixation under ChIP conditions

250pg each of in vitro transcribed MT-Tcf1-FLAG mRNA, MT-Lef1-FLAG mRNA, MT-Tcf3-FLAG mRNA and MT-Tcf4a-FLAG mRNA were injected into 4-cell stage embryos, cultured until the blastula stage (NF9) and fixed for ChIP-type IP (see 3.7.7). 3eeq Tcf-Mix injected embryo lysate (X) or untreated embryos (UI) were used for the “ChIP”-type IPs with LEF 3D4, LEF 5F12, Tcf1 2F4 and Tcf3 3E5. ProteinG-sepharose was blocked for Lef1 and Tcf1 IPs in the presence of 0.05% Tween20 (beads Lef1+Tcf1), for Tcf3 without Tween20 (beads Tcf3). The proteins were bound to the antibodies with or

with out the presence of 10U DNaseI (Roche). 33% of the inputs were loaded on the SDS-PAGE. The immunoblotting was performed with αMyc antibody 9E10. IgG-HC:

IgG heavy chain. For the analysis of DNaseI treatment, the chromatin was decrosslinked; the DNA was purified and analyzed on a 1% agarose gel.

A direct interaction of Lef/Tcf protein was not reported so far, although Lef/Tcf binding sites frequently appear as multimers on promoter elements like the siamois promoter (Brannon et al., 1997). Still, it is possible that Lef/Tcf proteins interact in someway directly or indirectly. The co-precipitation under formaldehyde-fixed conditions was further analyzed with the bimolecular fluorescence complementation technique (BiFC) (Hu et al., 2002). However, all attempts to demonstrate an interaction between Lef/Tcf proteins in vivo were fruitless, although the positive controls bJun fused to the N-terminal part of YFP and bFos fused to the C-terminal part of YFP showed the YFP fluorescent signal.

From these results, I conclude that the αLef1 LEF 3D4 and LEF 5F12

and αTcf1 Tcf1 2F4 are highly specific and do not cross-react with the other

Lef/Tcf family members. The Tcf3 antibody Tcf3 3E5 was not able to precipitate its antigen under native conditions. Unexpected, the αTcf1, αLef1 and αTcf3

antibodies precipitated their own antigen. This precipitation is very specific under unfixed condition. The co-precipitation their family members under formaldehyde-fixed ChIP conditions in a DNaseI-treatment independent manner suggests that they are some uncharacterized parts of multimeric complexes, although they do not interact directly.