Article Figures & Tables
Figures
- Fig. 1.
Phylogenetic tree of BH3-only proteins. Sequences found for zebrafish, Xenopus, human and axolotl were translated into amino acid sequences and aligned using ClustalW2 and T-coffee. A tree was generated and visualized. The scale bar indicates amino acid changes.
- Fig. 2.
Alignment of BH3 domains. (A) BH3 consensus sequence as described by Day et al. (2008) is given in the first line. Amino acids identified as possible BH3 domains in putative axolotl orthologs are given below. Residues matching consensus are marked in yellow, while residues differing from the consensus are marked in orange. (B) WebLOGO plots based on the alignment of human, zebrafish, Xenopus and axolotl BH3 domains of Bim, Beclin and Bmf.
- Fig. 3.
RT-PCR analysis. cDNAs prepared from the indicated tissues were used for amplification of BH3-only sequences using the primers as given in Table 2.
- Fig. 4.
Western blot analysis. Whole axolotl organ lysates were subjected to western blotting followed by immunological detection of Bmf, Bid, Bnip-3, Bad and Actin.
- Fig. 5.
Expression of Bcl-2 family members in axolotl limb regeneration blastema. After reverse transcription of limb blastema derived RNA, gene-specific products were amplified based on the sequences retrieved from Sal-Site (http://www.ambystoma.org/) as indicated in Table 1.
- Fig. 6.
TUNEL staining of early limb regeneration stages. (A) Wound closure after 48 h. Apoptosis is stained in wound epithelium (WE), muscle (M) and periost near the amputation site. (B) Dedifferentiation stage (DT) after 5 days. Apoptosis is enhanced in the mesenchymal tissue beneath the thickened wound epidermis (apical epithelial cap, AEC). (C) In the mid-bud stage, only single apoptotic cells are observed in AEC and regeneration blastema (B).
- Fig. 7.
(A-C) Immunological detection of Bak (red fluorescence) in early limb regeneration stages. (D-F) Immunological detection of Bcl-2 (green fluorescence). Nuclei were counterstained with DAPI (blue fluorescence). (A) Single cells were found in the wound epithelium (WE) and adjacent muscle tissue (M) although a diffuse background staining is also found in the muscle tissue. (B) Bak is mainly expressed in the dedifferentiating tissue (DT), but single cells are also found in the apical epithelial cap (AEC) and muscle. (C) In the blastema (B) evenly distributed, single cells express Bak. In the AEC, most cells expressing Bak are found in the proximal cell layers. (D) Bcl-2 positive cells are regularly dispersed in WE and adjacent tissue. (E) Most Bcl-2 expressing cells are found in the DT layers next to the AEC. (F) Bcl-2 is expressed in the B and in the proximal layers of the AEC. (G,H) Tests with secondary antibodies.
- Fig. 8.
Co-localization of Bcl-2 and Bid. (A,B) In mid-bud stage, cells were stained for Bcl-2 expression (A; green fluorescence) and Bid expression (B; red fluorescence). (C) Nuclei were counterstained with DAPI (blue fluorescence). (D) Merged image. AEC, apical epithelial cap; B, blastema.
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