Figure 4. ISWI binds to RanGTP-induced asters/spindles and RanGTP-stabilized centrosomal asters. (A) GFP-ISWI is incorporated into poles and MTs of RanGTP-induced asters and spindles. A CSF extract containing 1.3 µM GFP-ISWI or GFP was incubated with RanQ69L and Cy3-tubulin at 20°C for 30 min. The samples were fixed and spun down to coverslips. Bar, 10 µm. (B) Endogenous ISWI localizes to the poles and MTs of the Ran asters/spindles. The RanGTP-nucleated asters and spindles were stained by anti–full-length ISWI antibody and Alexa 488–labeled anti–rabbit IgG (green). Bar, 10 µm. (C) GFP-ISWI is incorporated into RanGTP-stabilized centrosomal asters. A CSF extract containing 1.3 µM GFP-ISWI or GFP control was incubated with centrosomes, anti-TPX2 antibody, and Cy3-tubulin in the presence or absence of RanQ69L at 20°C for 30 min. Anti-TPX2 antibody inhibits RanGTP-dependent MT nucleation. The samples were fixed and spun down on coverslips. Bar, 20 µm.
Figure 4.

ISWI binds to RanGTP-induced asters/spindles and RanGTP-stabilized centrosomal asters. (A) GFP-ISWI is incorporated into poles and MTs of RanGTP-induced asters and spindles. A CSF extract containing 1.3 µM GFP-ISWI or GFP was incubated with RanQ69L and Cy3-tubulin at 20°C for 30 min. The samples were fixed and spun down to coverslips. Bar, 10 µm. (B) Endogenous ISWI localizes to the poles and MTs of the Ran asters/spindles. The RanGTP-nucleated asters and spindles were stained by anti–full-length ISWI antibody and Alexa 488–labeled anti–rabbit IgG (green). Bar, 10 µm. (C) GFP-ISWI is incorporated into RanGTP-stabilized centrosomal asters. A CSF extract containing 1.3 µM GFP-ISWI or GFP control was incubated with centrosomes, anti-TPX2 antibody, and Cy3-tubulin in the presence or absence of RanQ69L at 20°C for 30 min. Anti-TPX2 antibody inhibits RanGTP-dependent MT nucleation. The samples were fixed and spun down on coverslips. Bar, 20 µm.

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