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Figure 6.
Figure 6. Genomic organisation and transfection of muscle
ponsin. (a) Schematic drawing of the organisation of the human
ponsin gene. Each box represents an exon, alternatively spliced
exons are highlighted by grey shading and numbers (A1). The
second drawing illustrates which exons are represented in the
splice variant of ponsin cloned from skeletal muscle cells (A2).
The portions encoding sorbin homology regions (SoHo), the SH3
domains and a 278 aa long muscle-specific insertion in the C
terminus (sinuous line) are indicated. The resulting full-length
protein (Ps FL, A3) is shown subsequently. In addition,
schematic drawings of deletion constructs used for transient
transfection experiments are given: an N-terminal construct
containing the sorbin homology region (Ps N-Ex25, A4), a
construct compromising the entire C terminus (Ps SH3.1-C, A5)
and a construct that consists only of the three C-terminal SH3
domains, but lacks the insertion (Ps SH3.1-C ΔEx30,31, A6). (b)
Transient transfection of ponsin constructs into neonatal rat
cardiomyocytes. Cells were transfected using either the
GFP-tagged full-length muscle ponsin isoform (Ps FL, a–d), the
N-terminal construct (Ps N-Ex25, e–h), the C-terminal ponsin
construct (Ps SH3.1-C, i–m), the C-terminal ponsin construct
lacking the insertion (Ps SH3.1-C ΔEx30,31, n–q) or GFP alone
(r–u), respectively. At 48 h post transfection cells were
triple labeled for paxillin (b, f, k, o, s) and a marker for
intercalated disc structures (β-catenin; c, g, l, p, t). The
C-terminal constructs that contained the three SH3 domains (i
and n), co-localised with endogenous paxillin at cell–matrix
contacts. In contrast, the N-terminal ponsin construct (e) was
targeted predominantly to the nucleus. Note that paxillin was
not observed in intercalated disc structures as delineated by
β-catenin. Scale bar represents 10 μm.
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