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. 2015 Sep 28;210(7):1165–1184. doi: 10.1083/jcb.201506041

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© 2015 Leyme et al.

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 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — GIV promotes Akt activation upon integrin stimulation. (A) GIV depletion does not affect MDA-MB-231 cell adhesion to different ECM substrates. MDA-MB-231 control shRNA and GIV shRNA2 cells were seeded on plates coated with collagen I, fibronectin, laminin, vitronectin, Matrigel, or BSA (as negative control), and cell adhesion was determined 1 h later as described in Materials and methods. Results are depicted as mean ± SEM (error bars; n = 3). (B) Schematic representation of the protocol followed to monitor ECM-specific cell stimulation. Cells were lifted, kept in suspension for 1 h in serum-free media, and seeded on surfaces coated with different ECM components in the absence of serum. Cells were harvested at different time points after seeding for subsequent analyses. Under these conditions, the only stimulus for the cells is mediated through binding to the ECM. (C and D) GIV depletion impairs Akt activation upon integrin activation in MDA-MB-231 cells. MDA-MB-231 control shRNA and GIV shRNA2 cells were stimulated by collagen I (C) or Matrigel (D), as described in B. (C and D; top) Representative immunoblots for the time course of Akt activation (as measured by levels of pAkt) upon ECM stimulation in MDA-MB-231cells. (C and D; bottom) Quantification of Akt activation (as described in Materials and methods). Error bars represent mean ± SEM (n = 3; **, P < 0.01; ***, P < 0.001). (E) Exogenous GIV expression in MCF-7 cells is sufficient to enhance Akt activation in response to collagen I stimulation. MCF-7 cells stably expressing GIV (GIV WT) or an empty plasmid (Control) were stimulated with collagen I as described in B. Some cells were preincubated with PP2 (as indicated). (Left) Representative immunoblots for the time course of Akt activation (as measured by levels of pAkt) upon collagen I stimulation in MCF-7 cells. (Right) Quantification of Akt activation (as described in Materials and methods). Results are depicted as mean ± SEM (error bars; n = 3–7; ***, P < 0.001). (Inset) Expression of exogenous GIV in MCF-7 cells was verified by immunoblotting (IB) using the indicated antibodies.