MLL5 is a centrosomal protein. (A) MLL5 centrosomal localization. U2OS cells were immunostained for MLL5 (green) and γ-tubulin (red). DNA was counterstained with DAPI (blue). Bars, 10 µm. Insets show high-magnification (2.5×) images of the centrosome at interphase. (B) Sucrose gradient centrifugation isolating the centrosome components. Fractions with concentrations ranging from 40% to 60% were collected and detected by Western blotting (WB) with anti-MLL5 and anti–γ-tubulin antibodies. (C) Interaction between endogenous MLL5 and γ-tubulin. After synchronization to prometaphase by nocodazole treatment, U2OS cell lysates were immunoprecipitated with anti-MLL5 antibody or mouse IgG and detected by Western blotting with anti-MLL5 and anti–γ-tubulin antibodies. (D) Interaction between FLAG-MLL5 and γ-tubulin. 293T cells expressing FLAG-MLL5 were synchronized to prometaphase by nocodazole treatment before immunoprecipitation and Western blotting. (E) Schematic representation of truncated MLL5 mutants. PS, PHD/SET domain; CT, C-terminal domain. (F) The central domain of MLL5 binding to γ-tubulin. 293T cells expressing FLAG-MLL5-PS, FLAG-MLL5-CD, or FLAG-MLL5-CT were synchronized to prometaphase before immunoprecipitation and Western blotting. Arrowhead indicates a slow-migrating form of FLAG-MLL5-CD. Results in B–D and F are representative of at least three experimental repeats.