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First published online February 13, 2009
Journal of Experimental Biology 212, 668-672 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022681
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Characterization of amphioxus nebulin and its similarity to human nebulin

Akira Hanashima1, Kaoru Kubokawa2 and Sumiko Kimura1,*

1 Department of Biology, Graduate School of Science, Chiba University, Chiba 263-8522, Japan
2 Center for Advanced Marine Research, Ocean Research Institute, University of Tokyo, Tokyo 164-8639, Japan


Figure 1
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  		Fig. 1. Domain structures and primary structure of the nebulin cDNA C-terminal region in amphioxus striated muscle. (A) Domain structures. Thin black bars at the top show the size of each clone and the thicker bar below indicates the position of the Pc AmpN antigen. (B) Amino acid sequence of amphioxus nebulin. (C) Consensus sequences of amphioxus and human nebulin repeats.

 

Figure 2
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  		Fig. 2. Comparison of the C-terminal region of amphioxus and human nebulins. The domain structure and isoelectric point (pI) are presented according to Labeit and Kolmerer (Labeit and Kolmerer, 1995Go), and the I-Z border is presented according to Millevoi et al. (Millevoi et al., 1998Go). Amphioxus, amphioxus nebulin; Human, human nebulin.

 

Figure 3
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  		Fig. 3. SDS-PAGE and immunoblot detection of amphioxus striated muscle. (A) Antigen for amphioxus nebulin. M, molecular size markers; lane 1, Pc AmpN antigen. (B,C) Immunoblot detection of amphioxus striated muscle using Pc AmpN (thick bar in Fig. 1A). Electrophoresis was performed on 2–6% (B) or 2–15% (C) polyacrylamide gradient gels. M, molecular size marker; lane 1, amido black stains; lane 2, immunoblot detection by Pc AmpN. C, connectin; N, nebulin; MHC, myosin heavy chain.

 

Figure 4
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  		Fig. 4. Immunofluorescence microscopic observations of amphioxus striated muscle sarcomeres. (A) Phase-contrast image. (B,C) Immunostaining with Pc AmpN (B) and {alpha}-actinin (C) monoclonal antibodies. (D) Merged image of B and C. Arrowheads, Z-line. Scale bar, 10 µm.

 

Figure 5
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  		Fig. 5. Binding of amphioxus nebulin and F-actin examined by co-sedimentation. Actin, actin only; AN3–9, amphioxus nebulin repeats 3–9 dissolved in GST; AN3–9+Actin, amphioxus nebulin repeats 3–9 +actin; AN3–9+BSA, amphioxus nebulin repeats 3–9 +BSA. W, before centrifugation; S, supernatant after centrifugation; P, pellet after centrifugation. Electrophoresis was performed on a 12.5% polyacrylamide gel.

 

Figure 6
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  		Fig. 6. Binding of amphioxus nebulin and {alpha}-actinin, examined by a GST pull-down assay. Lane 1, molecular size marker; lane 2, {alpha}-actinin used for the pull-down assay; lanes 3–6, after the pull-down assay: lane 3, GST fusion protein for amphioxus nebulin repeats 3–9 –{alpha}-actinin; lane 4, GST only –{alpha}-actinin; lane 5, GST fusion protein for amphioxus nebulin repeats 3–9 +{alpha}-actinin; lane 6, GST only +{alpha}-actinin. GST–AN3–9GST, fusion protein for amphioxus nebulin repeats 3–9. Electrophoresis was performed on a 10% polyacrylamide gel.

 

Figure 7
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  		Fig. 7. Binding of amphioxus nebulin and connectin examined by a GST pull-down assay. Lane 1, molecular size marker; lane 2, rabbit skeletal muscle; lane 3, connectin used for the pull-down assay; lanes 4–7, after the pull-down assay: lane 4, GST–U–SH3 –connectin; lane 5, GST only –connectin; lane 6, GST–U–SH3 +connectin; lane 7, GST only +connectin. GST-U–SH3, GST fusion protein for amphioxus nebulin U–SH3 region. Electrophoresis was performed on a 2–15% polyacrylamide gel.

 

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© The Company of Biologists Ltd 2009