Nedd1 is required for MT anchoring. (A) Images of MT organization in control, DP-NγBD–, DP-CγBD–, or DP-CγBD + Nedd1KD–expressing cells under steady-state (nonperturbing) conditions. Insets show higher magnifications of cortical regions. Bars: (main) 10 µm; (insets) 2 µm. Note that cell junctions are acting as discrete MTOCs in DP-CγBD–expressing cells. (B) Quantification of the cortical MT-anchoring activity of cells expressing the DP fusion constructs. (C) Representative images of MT assembly assays using centrosomes purified from control and Nedd1 knockdown cells. Bar, 10 µm. (D) Quantification of MT aster area around control and Nedd1 knockdown centrosomes (n ≥ 23 centrosomes from two independent experiments). (E) Quantification of free (noncentrosomal) MTs nucleated by control and Nedd1 knockdown centrosomes (n = 3 independent experiments). (F) Representative images of control and Nedd1 KD cells at indicated time points after nocodazole washout. Insets show zoomed in centrosomes. Bars: (main) 10 µm; (insets) 1 µm. (G) Quantification of the ratio of α-tubulin intensity at the centrosome to intensity in the cytoplasm in control and Nedd1KD keratinocytes after nocodazole washout (n ≥ 146 centrosomes from three independent experiments). (H) Quantification of MT nucleation at the centrosome and cytoplasm after nocodazole washout in control and Nedd1 KD keratinocytes (n ≥ 146 centrosomes from three independent experiments). n.s., not significant; **, P < 0.01; ***, P < 0.001. Data are presented as mean ± SEM.