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. 2010 Oct 1;21(19):3317–3329. doi: 10.1091/mbc.E10-03-0245

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© 2010 by The American Society for Cell Biology

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. — Role of PAK4 in MCF-7 cell adhesion on VN. (A) Left, cell attachment of MCF-7 cells stably expressing Flag-PAK4 or Flag-BAP at different coating concentrations of VN was determined. Graph shows means of optical density ± 95% confidence intervals; n = 3 from one representative experiment. Right, the protein levels of endogenous PAK4 and stably expressed Flag-PAK4-WT or Flag-BAP (as a control) in MCF-7 cells were detected by IB using anti-PAK4 pAb (top panel), anti-Flag (M2) mAb (middle panel) with anti-actin mAb as loading control (bottom panel). (B) Left, cell attachment of MCF-7 cells transiently expressing PAK4-siRNA or control siRNA at different coating concentrations of VN was determined. Graph shows means of optical density ± 95% confidence intervals; n = 3 from one experiment. Right, PAK4 siRNA-mediated knockdown was determined by IB using tubulin as loading control. (C) Cell attachment was determined of MCF-7 stably expressing control shRNA and stable PAK4 shRNA expressing MCF-7 cells transiently transfected with EGFP (as a control) or EGFP-PAK4-AC285, 288GA (RNAi-resistant PAK4) at different coating concentrations of VN. Graph shows means of number of cells per field ± 95% confidence intervals; n = 3 from one experiment. (D) PAK4 shRNA-mediated knockdown and expression levels of EGFP and EGFP-PAK4 for cells used in C were determined by IB using actin as loading control. All experiments in Figure 2 were repeated at least three times with similar results. p values are indicated for statistically discernable differences compared with control (A and B) or to PAK4-shRNA cells (C) according to unpaired two-tailed t test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

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