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Z. Tang, D.L. Boyle, D. Lin, T.A. Nguyen, D.J. Takemoto; IGF-1 Effects on Gap Junctions in ARPE-19 Cells : Role of PKC-gamma . Invest. Ophthalmol. Vis. Sci. 2003;44(13):373.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify gap junctions in the retinal pigment epithelial cell line ARPE-19; To detect the effects of TPA and IGF-1 on ARPE-19 gap junctions. Methods:Whole cell lysates were extracted with lysis buffer (20mM Tris pH 7.5, 0.5 mM EDTA, 0.5mM EGTA, 0.5% Triton X-100, 25:g/ml aprotinin, 25:g/ml leupeptin) and used for western blot and immunoprecipitation to identity connexins present in ARPE-19 cells and to identify interactions between connexins and PKC's. ARPE-19 cells (90% confluency) were reacted with Cx46 antibody and secondary antibody Alexa568, gap junction plaques were counted using confocal microscopy. Cell gap junction activities were determined by scrape loading/dye transfer (SL/DT). Results:Cx26, Cx32, Cx40, Cx43 and Cx46 were present in ARPE-19 cells; TPA (200 ng/ml) treatment decreased gap junction activity in ARPE-19 cells compared to untreated cells; TPA(200ng/ml) treatment translocated PKC-α, PKC- γto the membrane. IGF-1(25 ng/ml) treatment increased Cx46 gap junction plaque number in ARPE-19 cells compared to untreated cells; TPA treatment enhanced interactions between Cx43 and PKC-γ. Conclusions: Results suggest that ARPE-19 gap junctions are regulated by IGF-1 through PKC's.
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