Figure 2. ZAKβ redistributes to nuclear domains and stress fibers after osmotic shock.

• A
  U2OS cells stably expressing Strep‐HA‐ZAKβ or ‐ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody, and counterstained with DAPI.
• B
  U2OS cells stably expressing ZAKβ‐GFP were treated with sorbitol (500 mM, 1 h), pre‐extracted, fixed, and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line.
• C
  As in (B) except that Hela cells were transfected with ZAKbeta‐GFP and immunostained with 53BP1 antibody.
• D
  Cells from (B) were imaged by live‐cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after the acquisition of the first frame.
• E
  As in (D), except that cells were co‐transfected with mCherry‐FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers.
• F
  Schematic of GFP‐tagged ZAKβ truncation constructs.
• G
  U2OS cells were transfected with constructs from (F) and imaged by live‐cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after the acquisition of the first frame.
• H
  U2OS and ΔZAK cells stably expressing the indicated Strep‐HA‐tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.

Data information: All scale bars, 10 μm.

Source data are available online for this figure.