Rab40b – Cullin5 regulates localization and dynamics of FA sites. (A) Representative images of control, FLAG-Rab40b, and FLAG-Rab40b-4A MDA-MB-231 cells fixed and stained with phalloidin–Alexa Fluor 594 (red), anti-paxillin (green, FA marker), and DAPI (blue). Insets highlight anti-paxillin–stained regions of interest. (B) WB analysis of cell lysates using anti-paxillin (top blot) and anti-tubulin (bottom blot) antibodies. Numbers shown are densitometry analysis from at least three separate experiments relative to tubulin and standardized to control levels. Control::FLAG-Rab40b-4A, P < 0.05. (C) Quantification of number of FAs per cell for control, FLAG-Rab40b, and FLAG-Rab40b-4A cells. n ≥ 20 cells per condition. (D) Quantification of FA size in control, FLAG-Rab40b, and FLAG-Rab40b-4A cells. n = 2,479 total adhesions analyzed. (E) Quantification of FA average life span in control and FLAG-Rab40b-4A cells. Average life span of FAs was calculated from time-lapse images, with three cells per condition. (F) Quantification of percentage of FAs within 2 µm of cell border in control and FLAG-Rab40b-4A cells.