Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Binding of amphioxus nebulin to actin, α-actinin and connectin examined by far-western blotting. (A) Fusion-protein regions used for far-western blotting of amphioxus nebulin. (B) Binding of amphioxus nebulin and F-actin. Molecular size marker, lane M; amido black staining of (AN3−9+GST), lane 1; treated with F-actin and then reacted with actin antibody, lane 2; treated without actin and then reacted with actin antibody (control), lane 3. GST3−9 is the GST fusion protein of amphioxus nebulin repeats 3−9. (C) Binding of amphioxus nebulin and α-actinin. Molecular size marker, lane M; treated with polyhistidine antibody, lane 1; treated with α-actinin and then reacted with α-actinin antibody, lane 2. Regions between neb1 and 2, between neb3 and 9, between neb10 and unique region, and between unique region and SH3 domains of amphioxus nebulin correspond to 1−2, 3−9, 10−U and U−SH3, respectively, which are histidine-tagged proteins. (D) Binding of amphioxus nebulin and connectin. Molecular size marker, lane M; amido black staining of (U−SH3+GST), lane 1; treated with connectin and then reacted with connectin antibody, lane 2; treated without connectin and then reacted with connectin antibody, lane 3 (control). U−SH3 is the GST fusion protein of amphioxus nebulin U−SH3. Method used to obtain the results shown in Fig. S1: GST-fusion proteins mixed with GST, and the bacteria with the His-tag proteins were dissolved in SDS sample buffer, subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 20 mg ml⁻¹ BSA in buffer (80 mmol l⁻¹ KCl, 2 mmol l⁻¹ MgCl2, 10 mmol l⁻¹ Hepes, 1 mmol l⁻¹ DTT and 2% Triton X-100, pH 7.3, for α-actinin or connectin) or 0.5% casein in buffer (2 mmol l⁻¹ Tris, 0.01% NaN₃, 0.1 mmol l⁻¹ CaCl₂ and 100 mmol l⁻¹ KCl, pH8.0, for F-actin) at 25°C for 1 h. The membranes were incubated with F-actin (0.3 mg ml⁻¹), α-actinin (10 µg ml⁻¹) or connectin (80 µg ml⁻¹) in each buffer at 25°C for 1.5 h. After washing two times with TBS for 10 min, binding was detected with anti-actin (Sigma A4700; Sigma-Aldrich Japan), anti α-actinin (Sigma A7811; Sigma-Aldrich Japan) or anti-connectin (Pc1200) (Kimura et al., 1992) antibody.

• Supplemental Figure S2 -

  Fig. S2. Binding of amphioxus nebulin repeat and BSA examined by GST pull-down assay. We examined the binding of amphioxus nebulin repeats 3−9 and BSA as a control for binding of nebulin repeats 3−9 and α-actinin, by a GST pull-down assay. Molecular size marker, lane 1; BSA used for pull-down assay, lane 2; and after pull-down assay, lanes 3 and 4. GST fusion protein for amphioxus nebulin repeats 3−9 + BSA, lane 3; GST fusion protein for amphioxus nebulin repeats 3−9 − BSA, lane 4. GST−AN3−9 is the GST fusion protein for amphioxus nebulin repeats 3−9. Electrophoresis was performed on a 10% polyacrylamide gel.