PKCalpha and PKCgamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line
| Autor: | L M, Wagner, D J, Takemoto |
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| Rok vydání: | 2001 |
| Předmět: |
Electrophoresis
Agar Gel Microscopy Confocal Protein Kinase C-alpha Reverse Transcriptase Polymerase Chain Reaction Blotting Western Gene Expression Cell Differentiation Epithelial Cells Transfection Crystallins Cell Line Isoenzymes Lens Crystalline Animals Electrophoresis Polyacrylamide Gel Microscopy Phase-Contrast RNA Messenger Rabbits Protein Kinase C DNA Primers |
| Zdroj: | Molecular vision. 7 |
| ISSN: | 1090-0535 |
| Popis: | The overexpression of PKCalpha or PKCgamma for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for alphaA-, alphaB-, beta-, and gamma-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development.Cells overexpressing PKCalpha or PKCgamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCalpha or PKCgamma antibodies along with either alphaA-, alphaB-, beta-, or gamma-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either alphaA-, alphaB-, beta-, or gamma-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 microg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis.alphaB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCalpha and PKCgamma overexpression. alphaA-crystallin was only detectable in lentoid bodies after PKCalpha or PKCgamma overexpression. RT-PCR was able to detect beta-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of beta-crystallin protein. No gamma-crystallin expression was noted in these lentoid bodies.Overexpression of PKCalpha or PKCgamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only alphaB-crystallin but alphaA- and beta-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell. |
| Databáze: | OpenAIRE |
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