Rab40b – Cullin5 affects individual cell migration. (A) WB analysis of lysates from MDA-MB-231 cells stably expressing WT FLAG-Rab40b or FLAG-Rab40b-4A. (B) Directionality of migrating cells derived from time-lapse analysis of control or FLAG-Rab40b-4A cells (see Videos 1 and 2) for cells that remained in frame for the duration of the experiment. Data are shown as means and SEM. n is the number of cells analyzed. (C and D) Control, FLAG-Rab40b, and FLAG-Rab40b-4A cells were plated on collagen-coated coverslips and then fixed and stained with phalloidin–Alexa Fluor 594. Zoomed regions of interest highlight differences in cytoskeletal architecture, and arrows point to stress fibers. (D) Quantification of cells with prominent stress fibers. n ≥ 100 cells per condition. (E) Chemotactic assays of control, FLAG-Rab40b, or FLAG-Rab40b-4A cells on collagen coating, plated in either low- or high-serum conditions. Results are of three separate runs, with at least three wells per condition per run. Left panel: Grouped scatter plot of relative migration over the entire time course. Right panel: Bar graph showing relative migration at 24 h. (F) Chemotactic assays of control or FLAG-Rab40b-4A cells plated on either collagen or fibronectin. Results are of three separate runs, with at least three wells per condition per run.