Figure 2.

Local application of tensile forces modulates the subcellular localization of kindlin-2. (A–E) HCF were incubated with fibronectin-coated magnetic microbeads followed by tensile force application using a magnet. (A) Total and microbead-associated cell fractions were collected and analyzed by immunoblotting with indicated molecular weights (glyceraldehyde 3-phosphate dehydrogenase = GAPDH) and (B,C) quantified from band intensities. Fibronectin was used as loading control for microbead-associated fractions. (D,E) HCF were fixed, permeabilized, stained for kindlin-2 (green) and lamin B (red) and levels of nuclear kindlin-2 (fluorescence intensity, appearing yellow in overlays) were quantified by image analysis. Z-stacks of confocal optical sections were projected into one plane at (D) low and (E) high magnification. Insets show kindlin-2 staining only, bottom images show optical z-section reconstruction along the indicted plane (blue line) (see also Supplementary Videos S1 and S2). Shown are mean values from at least three independent experiments (data points) ±SD (** p < 0.01, using one sample Student t-test). All scale bars: 20 µm.