beta -Trcp couples beta -catenin phosphorylation-degradation and regulates Xenopus axis formation -- Liu et al. 96 (11): 6273 Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for Liu et al. (1999) Proc. Natl. Acad. Sci. USA 96(11), 6273-6278.

Plasmids.
b-Trcp cDNA was amplified from Xenopus leavis oocyte cDNAs by PCR with the following two primers: 5¢-CGGGATCCGCCATGGAAGGATTTTCATGTTCTC-3¢ and 5¢-GGGCTCGAGTTATCTGGAGATGTACGTGTA-3¢. The PCR product was subcloned into NcoI and XhoI sites of CS2+MT vector to generate CS2+MT-b-Trcp^myc. CS2+MT-b-TrcpDWD^myc was generated by deleting the EcoRI-XhoI fragment from CS2+MT-b-Trcp^myc. b-TrcpDF was amplified by 5¢-GGGCCATGGTGACCTCTGATGGAATGCTC-3¢ and 5¢-GGGCTCGAGTTATCTGGAGATGTACGTGTA-3¢, and was cloned into NcoI and XhoI sites of CS2+MT vector to generate CS2+MT-b-TrcpDF^myc. CS2+b-Trcp, CS2+b-TrcpDF, and CS2+b-TrcpDWD (for non-tagged b-Trcp and the mutants) were generated by deleting the BamHI-NcoI fragment from the myc-tagged versions.

b-catenin (S ® A) harbors identical mutations as described (1) and was generated by two PCR steps. The first step used the following two primers, with SP36T-b-catenin as the template: 5¢-TCTTACCTGGATGCTGGGATTCATGCTGGCGCCACCGCCACAGCACCAGCTTTGAGTGGC-3¢ and 5¢-CGTCATTAAGCAGTTTCG-3¢. The second step used the SP6 primer and the above PCR product as primers to amplify SP36T-b-catenin. The final PCR product was cut with NcoI and XhoI and used to replace the corresponding fragment in SP36T-b-catenin to generate SP36T-b-catenin (S ® A). CS2-b-catenin^Flag, which contains two copies of the Flag epitope at the b-catenin carboxyl terminus, was generated in the following way. Two primers, 5¢-GGACTAGTTACTTGTCGTCGTCGTCCTTGTAGTCGGGCTTGTCGTCGTCGTCCTTGTAGT-3¢ and 5¢-GTCAGTTGAGTTGACCAG-3¢, were used to amplify SP36T-b-catenin. The PCR product was cut with BstEII and SpeI, and was ligated together with the NcoI and BstEII fragment of b-catenin into the NcoI and XbaI sites of a modified CS2+ vector, generating CS2+b-catenin^Flag.

b-catenin or b-catenin (S ® A) cDNA was isolated from the above SP36T plasmids by NcoI and EcoRI digestion and inserted into the NcoI and EcoRI sites of a modified pGEX vector. pGEX-b-catenin-N1 and -N2 were generated by deleting the coding sequence 3¢ to the unique NarI site and the unique XhoI site, respectively (by deleting the NarI-NotI fragment and the XhoI-NotI fragment). Two primers, 5¢-GCAGCCATGGGCCACTGGCAGCAGCAGTC-3¢ and 5¢-CGGAATTCAATCTTCATCCTCTGGGTTTCC-3¢, were used to amplify the b-catenin-N3 fragment. The PCR product was cut with NcoI and EcoRI and subcloned into the pGEX vector, giving rise to pGEX-b-catenin-N3.

Axin^Flag, encoding an Axin molecule with an amino-terminal Flag epitope, was generated by site-directed mutagenesis according to the manufacturer’s instructions (Muta-Gene T7 Kit, Bio-Rad). Two mutagenesis primers were used: 5¢-AGGGTGCAGCGCTATCGATGCCACCATGGACTACAAAGACGATGACGACAAGCAGAGTCC-3¢ to introduce a ClaI site 5¢ to the Flag epitope in frame with the initiation ATG codon, and 5¢-AAGGTGGACTGATGATATCGCAGCACACCC-3¢ to introduce an EcoRV site 3¢ to the STOP codon. The original myc tag sequence and the 3¢ untranslated sequence in CS2+Axin (2) were removed by deleting both the ClaI-ClaI fragment and the EcoRV-SnaBI fragment. This gave rise to pCS2+Axin^Flag.

1. Yost, C., Torres, M., Miller, R. R., Huang, E., Kimelman, D. & Moon, R. T. (1996) Genes Dev. 10, 1443-1454.

2. Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., Lee, J. J., Tilghman, S. M., Gumbiner, B. M. & Costantini, F. (1997) Cell 90, 181-192.