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. 2016 Sep 26;214(7):859–873. doi: 10.1083/jcb.201601063

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© 2016 Binamé et al.

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Figure 2. — Identification of a domain targeting p190A to cell protrusions. (A) A region of p190A containing the two last FF motifs and the MD (2F-MD, 380–1,060 aa) was generated (see Table S3). Huh7 cells were transfected with HA-2F-MD, fixed, and stained for HA (green) and F-actin (red). Bottom panels show membrane outgrowth at higher magnification. Arrowheads show cell edge where p190A constructs and F-actin colocalize. (B) Schematic representation of N and C terminus truncations of 2F-MD. (C) Immunostaining of Huh7 cells transfected with HA-2F-MD3, fixed, and stained as in A. (D) Quantification of the number of cells presenting a localization of p190A constructs at actin-rich edges. Values are expressed as the mean ± SEM (n > 200 for each construct; three to four independent experiments). ****, P < 0.0001 when compared with p190AWT condition, by ANOVA followed by Tukey’s multiple-comparison test. (E) Huh7 cells were transfected with GFP or GFP-PLS, fixed, and stained for actin. Arrowheads point out colocalization of p190A constructs and F-actin. (F) Quantification of cells showing localization of GFP or GFP-PLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 360; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001. (G) Schematic representation of the p190AΔPLS protein compared with the full-length p190A protein (p190AWT). (H) Western blot analysis of Huh7 cells transiently expressing the recombinant proteins HA-p190AWT and HA-p190AΔPLS. (I) Huh7 cells were transfected with HA-p190AWT or HA-p190AΔPLS, fixed, and immunostained for HA (green) and F-actin (red). Arrowheads show actin-rich edges with construct localization; * indicates the cytoplasmic localization of p190AΔPLS. (J) Quantification of (I), indicating percentage of cells showing HA-p190AWT or HA-p190AΔPLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 715; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001.