Hook2 regulates anchoring of the centrosomes to the NE. (A) HeLa cell lysates treated with indicated siRNA for 36 h were IB for Hook2 for assessing the knockdown efficiency, and α-tubulin was used as the loading control. (B) Representative images showing centrosome detachment from the nucleus of HeLa cells upon depletion of dynein, dynactin, and Hook2. Centrosomes are stained with γ-tubulin, MTs with α-tubulin, and nucleus with DAPI. Bars, upper images, 10 µm; lower zoomed insets, 2 µm. (C) Quantification of centrosome–NE distance in HeLa cells 36 h after siRNA transfections (n = 3; 150 centrosomes/experiment). (D) Western blot analysis with anti-HA and anti-Hook2 antibodies to confirm Hook2 knockdown and expression of siRNA-resistant constructs of Hook2 (WT and dynein binding-defective mutants) in control and Hook2 siRNA-treated HeLa cells. α-Gubulin was used as a loading control. (E) Representative images of rescue in centrosome attachment upon expression of siRNA-resistant Hook2 (WT) in Hook2-depleted HeLa cells but not with dynein-defective mutants. Bars, 2 µm. (F) Quantification of rescue in centrosome–NE distance as described in E (n = 3; 100 centrosomes/experiment). Data represent mean ± SD (ns, not significant; ***, P < 0.001; Student’s t test).