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Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

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Chicken gizzard homogenates were separated by 10%  SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the recombinant 1E12 antibody. Adult  chicken gizzard was extracted in TBS alone (lane a) or TBS/6 M  urea (lane b). The recombinant 1E12 antibody was reactive with a  single band, Mr = 100,000, using both mild and stringent conditions. We observed no bands in the 40-kD migration position.  In addition, the sFv immunoreagent recognized purified gizzard  α-actinin (lane c). A control sample of chicken serum proteins  showed no immunoreactive bands (lane d).
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Figure 5: Chicken gizzard homogenates were separated by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the recombinant 1E12 antibody. Adult chicken gizzard was extracted in TBS alone (lane a) or TBS/6 M urea (lane b). The recombinant 1E12 antibody was reactive with a single band, Mr = 100,000, using both mild and stringent conditions. We observed no bands in the 40-kD migration position. In addition, the sFv immunoreagent recognized purified gizzard α-actinin (lane c). A control sample of chicken serum proteins showed no immunoreactive bands (lane d).

Mentions: To address the complication that some immunoblots showed the variable doublet at 40 kD, and to obtain an immunoreagent that was easier to use than the parent IgM molecule, we took advantage of recombinant protein technology to produce a single chain antibody (sFv) with 1E12 specificity (see Materials and Methods). Using the sFv immunoreagent, crude chicken gizzard extracts were analyzed by immunoblotting (Fig. 5). Polypeptides in lanes a–d were separated by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the sFv 1E12. Adult chicken gizzard was extracted in TBS (Fig. 5, lane a) or TBS containing 6 M urea (lane b). A single, strongly immunoreactive band, Mr = 100,000, was observed in the crude gizzard homogenates extracted under both mild and stringent conditions. No bands at the 40-kD migration position were detected with the sFv immunoreagent. As shown in lane c, the sFv 1E12 was also immunoreactive with homogeneously pure chicken gizzard α-actinin. In contrast, a control sample containing chicken serum proteins showed no reactive bands (lane d). Also, neither the secondary (anti-myc mAb) nor the tertiary antibodies (goat anti–mouse IgG), used for this immunoblot, exhibited cross-reactivity with the chicken gizzard extracts or the purified α-actinin (data not shown).


Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

Chicken gizzard homogenates were separated by 10%  SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the recombinant 1E12 antibody. Adult  chicken gizzard was extracted in TBS alone (lane a) or TBS/6 M  urea (lane b). The recombinant 1E12 antibody was reactive with a  single band, Mr = 100,000, using both mild and stringent conditions. We observed no bands in the 40-kD migration position.  In addition, the sFv immunoreagent recognized purified gizzard  α-actinin (lane c). A control sample of chicken serum proteins  showed no immunoreactive bands (lane d).
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Related In: Results  -  Collection

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Figure 5: Chicken gizzard homogenates were separated by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the recombinant 1E12 antibody. Adult chicken gizzard was extracted in TBS alone (lane a) or TBS/6 M urea (lane b). The recombinant 1E12 antibody was reactive with a single band, Mr = 100,000, using both mild and stringent conditions. We observed no bands in the 40-kD migration position. In addition, the sFv immunoreagent recognized purified gizzard α-actinin (lane c). A control sample of chicken serum proteins showed no immunoreactive bands (lane d).
Mentions: To address the complication that some immunoblots showed the variable doublet at 40 kD, and to obtain an immunoreagent that was easier to use than the parent IgM molecule, we took advantage of recombinant protein technology to produce a single chain antibody (sFv) with 1E12 specificity (see Materials and Methods). Using the sFv immunoreagent, crude chicken gizzard extracts were analyzed by immunoblotting (Fig. 5). Polypeptides in lanes a–d were separated by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the sFv 1E12. Adult chicken gizzard was extracted in TBS (Fig. 5, lane a) or TBS containing 6 M urea (lane b). A single, strongly immunoreactive band, Mr = 100,000, was observed in the crude gizzard homogenates extracted under both mild and stringent conditions. No bands at the 40-kD migration position were detected with the sFv immunoreagent. As shown in lane c, the sFv 1E12 was also immunoreactive with homogeneously pure chicken gizzard α-actinin. In contrast, a control sample containing chicken serum proteins showed no reactive bands (lane d). Also, neither the secondary (anti-myc mAb) nor the tertiary antibodies (goat anti–mouse IgG), used for this immunoblot, exhibited cross-reactivity with the chicken gizzard extracts or the purified α-actinin (data not shown).

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

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