MISP associates with p150^glued and regulates its distribution at the cortex. (A) p150^glued was immunoprecipitated from nocodazole-blocked HeLa cells and analyzed by Western blot with indicated antibodies. Asterisk, unspecific recognized band in the control mouse IgG immunoprecipitate. (B) MTs were pelleted by high-speed centrifugation after nocodazole or taxol incubation. Input (I), supernatant (S), and the pellet (P) were analyzed by Western blot with indicated antibodies. (C) Z-stack images (0.4 µm per stack) of p150^glued in metaphase HeLa cells depleted with MISP or control siRNA after cytoplasmic extraction. Right panel shows images with x-z projection. Arrowheads show the cortical localized p150^glued. (D) Quantification of the percentage of metaphase cells with cortical accentuated and distributed p150^glued; n = 50 cells each from three independent experiments. (E) HeLa cells transfected with control or MISP siRNA, or MISP siRNA under expression of GFP or siRNA-resistant versions of GFP-MISP WT/6DP/7AP/9AC were stained for p150^glued. Representative images with z-projection are shown. Arrowheads indicate the cortical localized p150^glued. (F) Quantification of the percentage of metaphase cells with cortical accentuated and distributed p150^glued. n = 50 cells each from three independent experiments. Bars: (C and E) 10 µm. Error bars in D and F represent SD. Student’s t test was used to calculate p-value for comparison of control and experimental samples.