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. 2013 Apr 1;24(7):995–1006. doi: 10.1091/mbc.E12-10-0723

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© 2013 Bliss et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Lasp-2 interacts with vinculin. (A) Lasp-2 (green) colocalizes with vinculin (red) at focal adhesions. HEK 293 cells were transfected with GFP–lasp-2 and stained for vinculin. Areas of colocalization are highlighted with arrows. Scale bar, 10 μm. (B) For analysis with the yeast two-hybrid (Y2H) system, yeast strain AH109 was cotransfected with a bait coding for lasp-2 and a prey coding for vinculin. Various vinculin prey fragments were then cotransformed with lasp-2, and the binding site for lasp-2 on vinculin was determined to be within the vinculin-tail. (C) Also using the Y2H system, the binding site for vinculin on lasp-2 was found to be the SH3 domain of lasp-2 using different lasp-2 bait constructs. (D) Lasp-2 binds to vinculin-tail in a saturable manner in ELISA. A 10-pmol amount of vinculin-tail or His-peptide (negative control) was immobilized on microtiter plates and incubated with increasing amounts of lasp-2. Bound lasp-2 was detected with an anti–lasp-2 antibody. Lasp-2 bound to vinculin-tail but much less to His-peptide. (E) Lasp-2 coimmunoprecipitates with vinculin. Endogenous vinculin was immunoprecipitated from HEK 293 cell lysates expressing either GFP or GFP–lasp-2. GFP–lasp-2 coimmunoprecipitated with vinculin.