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. 2012 Dec 1;23(23):4647–4661. doi: 10.1091/mbc.E12-04-0315

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© 2012 Koizumi 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 4: — RhoD is activated by FGF stimulation and required for FGF-induced protrusion formation. (A) Activation of RhoD by FGF stimulation. Activation of RhoD was analyzed by a pulldown assay with GST–mDia3C(GBD) and lysates of Myc–RhoD(wt)-transfected 10T1/2 cells stimulated with FGF2/4/8. Myc–RhoD was detected by immunoblotting with the anti-Myc mAb. (B) The degree of RhoD activation by FGF stimulation in the analysis of (A). The values are mean ± SD of three independent experiments. (C) Specific suppression of RhoD expression by RhoD siRNAs. The 10T1/2 cells were transfected with RhoD Stealth siRNA(1) or (2). The levels of endogenous RhoD and RhoF mRNAs were analyzed 48 h after the transfection by real-time PCR. The values are normalized with β-actin mRNA levels. ***, p < 0.001; #, p = 0.095; ##, p = 0.36 (not significant) by t test compared with control siRNA transfection. (D) Requirement of RhoD for FGF8-induced protrusion formation. The 10T1/2 cells were first transfected with Stealth siRNAs and 48 h later with EGFP–RhoD(wt) or siRNA(2)-resistant EGFP–RhoD. The cells were stimulated for ∼60 min with FGF8 48 h after the RhoD transfection, and the protrusion length was analyzed. n > 390. *, p < 0.0001 by t test.