Rac1 controls localization and function of FMNL2 at the cell–cell interface. (A) Western blot of dox-inducible GFP-Rac1 expression in MCF10A. (B) MCF10A cells expressing GFP or GFP-Rac1-L61 were seeded in 3D and stained for F-actin. (C) Confocal image of MCF10A cells expressing mCherry-Rac1-L61 and FMNL2-GFP in 3D. (D) Immunoprecipitations (IP) of endogenous E-Cadherin were obtained from MCF10A cells induced to express GFP, GFP-Rac1-L61, or GFP-Rac1-N17. Precipitates were equally divided to test for FMNL2 or β-catenin by immunoblotting. An unspecific mouse IgG was used as a control. (E) GST pull-down assays showing an interaction between purified GST-FMNL2 GBD+FH3 and GFP-Rac1-L61 and GFP–RhoC-V14 expressed in HEK cells. (F) Western blot demonstrating Rac1 siRNA efficiency in MCF10A cells. (G) Confocal image of MCF10A FMNL2-GFP–expressing cells transfected with the indicated siRNAs and stained for F-actin. (H) Quantification of intensity ratios of F-actin or FMNL2-GFP based on line scans of confocal images as shown in G. (I) FRAP analysis in MCF10A GFP-actin cells treated with the indicated siRNAs. Expression of mCherry-FMNL2ΔDAD rescues the effect of Rac1 silencing. (J) Statistical analysis of I. Error bars indicate SEM. *, P ≤ 0.05.