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Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody.

Lee M, Rhee K - Endocrinol Metab (Seoul) (2014)

Bottom Line: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.Ninein signals were significantly impaired in CPAP-depleted cells.The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis.

Methods: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.

Results: Ninein signals were significantly impaired in CPAP-depleted cells.

Conclusion: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

No MeSH data available.


Related in: MedlinePlus

Generation of a ninein polyclonal antibody. (A) Ninein is a 2096-chain amino acid protein. The 381-689 fragment of ninein (solid bar) was used to generate the ninein antibody. (B) HeLa lysates were subjected to immunoblot analysis using the affinity-purified ninein antibody. The estimated size of ninein is approximately 240 kDa.
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Figure 1: Generation of a ninein polyclonal antibody. (A) Ninein is a 2096-chain amino acid protein. The 381-689 fragment of ninein (solid bar) was used to generate the ninein antibody. (B) HeLa lysates were subjected to immunoblot analysis using the affinity-purified ninein antibody. The estimated size of ninein is approximately 240 kDa.

Mentions: We began our study by generating a ninein-specific polyclonal antibody. Ninein is a large protein with 2096 amino acid residues (Fig. 1A). During antigen preparation, a ninein fragment (381-689 amino acid residues) was fused with the glutatione S-transferase (GST) tag and expressed in bacteria (Fig. 1A). Affinity-purified rabbit antiserum was used for immunoblot analysis. As reported previously, the ninein-specific band of the 240 kDa protein was detected in HeLa cell lysates (Fig. 1B) [7,8]. Accordingly, we used this antibody for immunostaining analysis of ninein in cultured cells.


Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody.

Lee M, Rhee K - Endocrinol Metab (Seoul) (2014)

Generation of a ninein polyclonal antibody. (A) Ninein is a 2096-chain amino acid protein. The 381-689 fragment of ninein (solid bar) was used to generate the ninein antibody. (B) HeLa lysates were subjected to immunoblot analysis using the affinity-purified ninein antibody. The estimated size of ninein is approximately 240 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384682&req=5

Figure 1: Generation of a ninein polyclonal antibody. (A) Ninein is a 2096-chain amino acid protein. The 381-689 fragment of ninein (solid bar) was used to generate the ninein antibody. (B) HeLa lysates were subjected to immunoblot analysis using the affinity-purified ninein antibody. The estimated size of ninein is approximately 240 kDa.
Mentions: We began our study by generating a ninein-specific polyclonal antibody. Ninein is a large protein with 2096 amino acid residues (Fig. 1A). During antigen preparation, a ninein fragment (381-689 amino acid residues) was fused with the glutatione S-transferase (GST) tag and expressed in bacteria (Fig. 1A). Affinity-purified rabbit antiserum was used for immunoblot analysis. As reported previously, the ninein-specific band of the 240 kDa protein was detected in HeLa cell lysates (Fig. 1B) [7,8]. Accordingly, we used this antibody for immunostaining analysis of ninein in cultured cells.

Bottom Line: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.Ninein signals were significantly impaired in CPAP-depleted cells.The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis.

Methods: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.

Results: Ninein signals were significantly impaired in CPAP-depleted cells.

Conclusion: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

No MeSH data available.


Related in: MedlinePlus