JCS02825 Supplementary Material

Files in this Data Supplement:

• Supplemental Table (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. Alignment of murine myopalladin with palladin isoforms. Protein sequences for myopalladin and palladin isoforms were derived from published NCBI/GenBank murine nucleotide sequences (XM483911.1 for myopalladin, AK052489 for 200 kDa palladin, AK031696 for 140 kDa palladin and the partial transcript BQ927960 for the N-terminus of 90 kDa palladin). The IgC2 domain organizations of palladin and myopalladin are highly conserved. This is especially evident for the 200 kDa isoform of palladin, which like myopalladin is highly expressed in striated muscle. In addition to the similarity of IgC2 domain sequence and organization, the nebulin/lasp-1 binding motif is also conserved between myopalladin and the 140 kDa and 200 kDa palladin isoforms.

• Supplemental Figure 2 -

  Fig. S2. The a-4IgNT polyclonal antibody does not cross-react with myopalladin. Heart, muscle and kidney lysates from a postnatal day eight mouse were immunoblotted with a polyclonal antibody developed against the N-terminus of myopalladin (provided by Siegfried Labeit, Universitätsklinikum Mannheim, Germany) or with the a-4IgNT antibody that recognizes 140 kDa and 200 kDa palladin. A band corresponding to myopalladin was detected with a-myopalladin in heart, but not in muscle or kidney (left panel). The 4IgNT antibody detected only the 200 kDa palladin isoform in heart and muscle, whereas the 140 kDa isoform is detected in kidney. A band approximating myopalladin was not detected with a-4IgNT in heart. Also, immunoreactive bands for a-myopalladin do not match immunoreactive bands for a-4IgNT. Therefore, there is no appreciable cross-reactivity between the a-4IgNT palladin antibody and myopalladin.  

• Supplemental Figure 3 -

  Fig. S3. Examples of bundled F-actin phenotypes observed in transfected COS-7 cells. Quantitation of bundling was facilitated by the categorization of observed phenotypes into one of three categories (small bundles, large bundles and stars). Region #1 is typical of the classification ‘small bundles’ and region #2 is typical of ‘large bundles’, which are more compact, thicker and brighter. The F-actin ‘star-like structures’ that form in 140 kDa palladin transfected COS-7 cells are distinct in both their compaction and symmetry, as shown in region #3.

• Supplemental Figure 4 -

  Fig. S4. Western blots of control siRNA and 140 kDa palladin siRNA transfected HeLa cells. The 140 kDa palladin siRNA sequence specifically silences this larger isoform and does not affect the expression of the 90 kDa isoform. The 90 kDa isoform was detected with the 1E6 monoclonal antibody. The 140 kDa isoform was detected with the a-4IgNT antibody.