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Fig. 1. EHD1-knockdown affects 
1 integrin transport and localization in HeLa cells. HeLa cells on plates (A,B) or coverslips (C,D,F) were mock-treated (A,B,C,F) or treated with EHD1-RNAi (A,B,D) for 48 hours. RNAi efficacy was determined following sample calibration for protein content, and immunoblotting for EHD1 (A; top panel) or actin control (A; bottom panel). Recycling of anti-1-integrin–1-integrin complexes to the plasma membrane in HeLa cells was quantitatively measured by a flow cytometry recycling assay using mouse anti-human 12G10 antibodies that preferentially recognize ligand-bound 1 integrins (MCA2028; Serotec) (B). The numbers denoted in the graph represent mean levels of 1 integrin fluorescence (from 10,000 cells) reappearing at the cell surface because of recycling. A similar pulse-strip-chase experiment was done in HeLa cells on coverslips to follow the internalization and subcellular distribution of 1 integrins upon EHD1-RNAi treatment (C compared with D). LSM 5 Pascale software was used to quantitatively determine the mean level of remaining, non-recycled internal 1 integrins by measuring the mean level of fluorescence per cell (E). Mock cells (n=100) yielded a mean of 475 with a standard deviation of 121, whereas EHD1-RNAi-treated cells (n=82) yielded a mean of 1112 with a standard deviation of 256. One-tailed Student's t-tests showed significance at P<0.0001. HeLa cells transfected with GFP-EHD1 were pulsed for 1 hour with 12G10 anti-1 integrin antibodies and visualized by confocal microscopy (F). Serial z-sections were obtained every 0.4 µm, and the crosshairs and arrows depict common membrane structures on a representative micrograph containing both EHD1 (green) and 1 integrins (red). Bar, 10 µm.
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