Abstract
Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.
| Original language | English |
|---|---|
| Pages (from-to) | 742-753 |
| Number of pages | 12 |
| Journal | Cell Biology International |
| Volume | 29 |
| Issue number | 9 |
| DOIs | |
| Publication status | Published - Sep 2005 |
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Keywords
- Antibodies
- Differentiation
- Myotonic dystrophy protein kinase
- Skeletal muscle cells
ASJC Scopus subject areas
- Cell Biology
Cite this
Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody : Possible role of the protein in cytoskeleton rearrangements during differentiation. / Beffy, Pascale; Barsanti, Cristina; Del Carratore, Renata; Simi, Silvana; Benedetti, Pier Alberto; Benzi, Luca; Prelle, Alessandro; Ciscato, Patrizia; Simili, Marcella.
In: Cell Biology International, Vol. 29, No. 9, 09.2005, p. 742-753.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody
T2 - Possible role of the protein in cytoskeleton rearrangements during differentiation
AU - Beffy, Pascale
AU - Barsanti, Cristina
AU - Del Carratore, Renata
AU - Simi, Silvana
AU - Benedetti, Pier Alberto
AU - Benzi, Luca
AU - Prelle, Alessandro
AU - Ciscato, Patrizia
AU - Simili, Marcella
PY - 2005/9
Y1 - 2005/9
N2 - Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.
AB - Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.
KW - Antibodies
KW - Differentiation
KW - Myotonic dystrophy protein kinase
KW - Skeletal muscle cells
UR - http://www.scopus.com/inward/record.url?scp=27644580746&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=27644580746&partnerID=8YFLogxK
U2 - 10.1016/j.cellbi.2005.05.011
DO - 10.1016/j.cellbi.2005.05.011
M3 - Article
C2 - 16099181
AN - SCOPUS:27644580746
VL - 29
SP - 742
EP - 753
JO - Cell Biology International
JF - Cell Biology International
SN - 1065-6995
IS - 9
ER -