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. 1999 Nov 15;147(4):879–890. doi: 10.1083/jcb.147.4.879

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© 1999 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9905059.f6ab.jpg — In vivo association of YAP65 and EBP50. (A) Whole cell lysates from wild-type 16HBE14o− cells were prepared in RIPA buffer and ∼300 μg of total protein was immunoprecipitated using EBP50-specific antisera (Ab) or normal rabbit IgG. Bound proteins were extensively washed in RIPA buffer, eluted, electrophoresed on 10% SDS-PAGE and blotted with rabbit anti-YAP65 (1:1,000) or mouse anti-ezrin (1:1,000) antisera. Input: 10% or 20% of the material added into each binding reaction. (B) Lysates from 16HBE14o− cells stably expressing GFP-YAP65 or GFP-YAP65/−4 (200 μg) were immunoprecipitated using EBP50-specific antisera as described in A. Bound proteins were electrophoresed on 10% SDS-PAGE and blotted with mouse anti-GFP (1:1,000). Input: 20% of the material added into each binding reaction. B, protein A beads alone; IgG, normal rabbit IgG; and Ab, rabbit anti-EBP50. (C) 16HBE14o− cells stably expressing GFP-YAP65 or GFP-YAP65/−4 were grown to confluence on transwell filters, fixed in 4% paraformaldehyde, and analyzed by confocal microscopy. At least two independent clones were analyzed for each cell line. Bars, 10 μm.

graphic file with name JCB9905059.f6c.jpg — In vivo association of YAP65 and EBP50. (A) Whole cell lysates from wild-type 16HBE14o− cells were prepared in RIPA buffer and ∼300 μg of total protein was immunoprecipitated using EBP50-specific antisera (Ab) or normal rabbit IgG. Bound proteins were extensively washed in RIPA buffer, eluted, electrophoresed on 10% SDS-PAGE and blotted with rabbit anti-YAP65 (1:1,000) or mouse anti-ezrin (1:1,000) antisera. Input: 10% or 20% of the material added into each binding reaction. (B) Lysates from 16HBE14o− cells stably expressing GFP-YAP65 or GFP-YAP65/−4 (200 μg) were immunoprecipitated using EBP50-specific antisera as described in A. Bound proteins were electrophoresed on 10% SDS-PAGE and blotted with mouse anti-GFP (1:1,000). Input: 20% of the material added into each binding reaction. B, protein A beads alone; IgG, normal rabbit IgG; and Ab, rabbit anti-EBP50. (C) 16HBE14o− cells stably expressing GFP-YAP65 or GFP-YAP65/−4 were grown to confluence on transwell filters, fixed in 4% paraformaldehyde, and analyzed by confocal microscopy. At least two independent clones were analyzed for each cell line. Bars, 10 μm.