Figure 3. BRISC is a MAP with cell... | Rockefeller University Press

$Figure 3. BRISC is a MAP with cell cycle–dependent localization. (A) The localization of ABRO1 in cell cycle. Exponential HeLa cells were fixed in cold methanol and immunostained for γ-tubulin (red) and ABRO1 (green); DNA was stained with DAPI. BRISC localizes at centrosomes, spindle poles, and midbody during mitosis. (B) K-fiber minus ends localization of ABRO1. Control (top), cold-treated (middle), and NUF2 siRNA–transfected (bottom) HeLa cells were fixed and stained with anti–α-tubulin (red) and anti-ABRO1 (green) antibodies. DNA was stained with DAPI. Bars, 5 µm. (C) MT sedimentation assay with purified recombinant FH–ABRO1. Purified FH–ABRO1 was mixed with in vitro–produced MTs (+MTs) or with buffer (−MTs) as a control. Subsequently, these samples were spun through a glycerol cushion, and the supernatant (S) and pellet (P) fractions were then analyzed for the presence of FH–ABRO1 by Western blotting. MTs produced in vitro with pure TAMRA rhodamine–labeled tubulin were spotted onto slides for visualization (right). FH–ABRO1 binds taxol-stabilized MTs in vitro. In parallel, the negative control experiment was performed with BSA. (D) MT sedimentation assay with HeLa mitotic lysate. Concentrated mitotic lysate was mixed with pure TAMRA rhodamine–labeled tubulin (+MT) or buffer (−MT) as a control, and the reactions were performed as described in Materials and methods. Subsequently, the samples were separated as described in C, and the sedimented MTs were analyzed for the presence of BRISC and NuMA by Western blotting. MTs produced in the mitotic lysate were spotted onto slides for visualization (bottom). BRISC and NuMA cosedimentation with MTs formed in the mitotic lysate.$

Figure 3.

BRISC is a MAP with cell cycle–dependent localization. (A) The localization of ABRO1 in cell cycle. Exponential HeLa cells were fixed in cold methanol and immunostained for γ-tubulin (red) and ABRO1 (green); DNA was stained with DAPI. BRISC localizes at centrosomes, spindle poles, and midbody during mitosis. (B) K-fiber minus ends localization of ABRO1. Control (top), cold-treated (middle), and NUF2 siRNA–transfected (bottom) HeLa cells were fixed and stained with anti–α-tubulin (red) and anti-ABRO1 (green) antibodies. DNA was stained with DAPI. Bars, 5 µm. (C) MT sedimentation assay with purified recombinant FH–ABRO1. Purified FH–ABRO1 was mixed with in vitro–produced MTs (+MTs) or with buffer (−MTs) as a control. Subsequently, these samples were spun through a glycerol cushion, and the supernatant (S) and pellet (P) fractions were then analyzed for the presence of FH–ABRO1 by Western blotting. MTs produced in vitro with pure TAMRA rhodamine–labeled tubulin were spotted onto slides for visualization (right). FH–ABRO1 binds taxol-stabilized MTs in vitro. In parallel, the negative control experiment was performed with BSA. (D) MT sedimentation assay with HeLa mitotic lysate. Concentrated mitotic lysate was mixed with pure TAMRA rhodamine–labeled tubulin (+MT) or buffer (−MT) as a control, and the reactions were performed as described in Materials and methods. Subsequently, the samples were separated as described in C, and the sedimented MTs were analyzed for the presence of BRISC and NuMA by Western blotting. MTs produced in the mitotic lysate were spotted onto slides for visualization (bottom). BRISC and NuMA cosedimentation with MTs formed in the mitotic lysate.

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