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Blood, Vol. 111, Issue 1, 142-149, January 1, 2008
Long-term, multilineage hematopoiesis occurs in the combined absence of β-catenin and  -catenin
Blood Jeannet et al.
111: 142
Supplemental material for: Jeannet et al
Files in this Data Supplement:
- Figure S1A & B. Immunoblot for β-catenin expression (PDF, 170 KB) -
Total cellular lysates from BM cells of the indicated genotypes were subjected to immunoblot analysis with mAbs to the COOH terminus of  -catenin (clone 14) and to tubulin (to ensure equal protein loading). (A) Wild type and heterozygous -catenin deleted BM cells of the two existing -catenin alleles (RK: Brault et al. Development 128:1253; JH: Huelsken et al. Cell 105:533). Numbers indicate the molecular weight in kDa. Note that a -catenin protein species of approximately 40-50 kDa is detected in BM cells of wt and both deleted -catenin alleles (indicated with an arrow). Expression of the shorter -catenin species was enhanced following cre-mediated recombination. These shorter proteins are not observed in non-hematopoietic tissues such as keratinocytes (B) (cf. Huelsken et al. Cell 105:533).
- Figure S1C. Schematic representation of the modified β-catenin (Ctnnb1) locus (PDF, 55 KB) -
Roman numbers depict exons (I-XVI) of the -catenin gene, and arabic numbers indicate the different armadillo (arm) repeats (1-12) encoded by the -catenin gene. Black triangles indicate loxP sites in intron 2 and 6 (Huelsken et al. Cell 105:533). In the floxed allele generated by the Kemler lab (Brault et al. Development 128:1253), the 5′ loxP site is located in intron 1 as shown by a gray triangle. Note that the targeting strategy of our floxed allele was designed such that after splicing of exon II to exon VII the ATG in exon II is out of frame. Primers used for genotyping flank the first loxP site in intron 2. A third primer in exon VII is used to detect the deleted allele. Cloning and expression studies show that the truncated -catenin protein species derive from the usage of two internal start codons (M328 and M363 in exons VII and VIII as indicated), which result in -catenin proteins starting in the middle of arm repeat 5 or at the beginning of arm repeat 6. These truncated -catenin proteins fail to interact with the NH2-terminal domain of TCF-1 (amino acids 1-117) in GST pull down experiments (data not shown). Deletion mutants had shown that the region of the -catenin protein required for binding to TCF/LEF is located in arm repeats 3-8 (indicated with a horizontal line; cf. Behrens et al. Nature 382:638, Graham et al. Cell 103:885, Poy et al. Nat. Struct. Biol. 8:1053). Exon III encodes the NH2-terminal domain (N), which contains the Wnt-regulated serine/threonine residues, which determine -catenin stability.
- Figure S1D & E Transcriptional activity of the truncated β-catenin protein species (PDF, 81 KB) -
(D) Wnt reporter activity as analyzed in 293Tcells in response to expression of TCF-1 in combination with -catenin, stabilized -catenin (S33A, S37A, T41A and S45A mutations) and the truncated -catenin cloned from mutant bone marrow. (E) Cell lysates used to measure Wnt reporter activity were also analyzed for -catenin expression by immunoblot. Arrows indicate the truncated -catenin protein species. Numbers indicate the molecular weight in kDa.
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