Figure 3.
Figure 3. Spindle targeting of astrin is mediated through the kinastrin-binding domain. (A and B) Myc-astrin (A) or Myc-kinastrin fragments (B) alone or in conjunction with FLAG-kinastrin (A) or FLAG-astrin (B) were transiently transfected into HeLa S3 cells for immunofluorescence analysis and HEK 293T cells for immune precipitations. HEK 293T cells were synchronized in mitosis by addition of the Eg5 inhibitor S-trityl-l-cysteine (STLC; 10 µM) for 14 h after 24 h of transfection. The cells were then harvested and lysed, and anti-Myc immunoprecipitations were performed. Western blots were probed as indicated. The asterisk indicates a degradation product. For immunofluorescence analysis, fixed HeLa cells were stained with antibodies to Myc, tubulin, and CREST serum. DNA was stained with DAPI. Numbers next to gel blots indicate molecular mass in kilodaltons. Bars, 10 µm.

Spindle targeting of astrin is mediated through the kinastrin-binding domain. (A and B) Myc-astrin (A) or Myc-kinastrin fragments (B) alone or in conjunction with FLAG-kinastrin (A) or FLAG-astrin (B) were transiently transfected into HeLa S3 cells for immunofluorescence analysis and HEK 293T cells for immune precipitations. HEK 293T cells were synchronized in mitosis by addition of the Eg5 inhibitor S-trityl-l-cysteine (STLC; 10 µM) for 14 h after 24 h of transfection. The cells were then harvested and lysed, and anti-Myc immunoprecipitations were performed. Western blots were probed as indicated. The asterisk indicates a degradation product. For immunofluorescence analysis, fixed HeLa cells were stained with antibodies to Myc, tubulin, and CREST serum. DNA was stained with DAPI. Numbers next to gel blots indicate molecular mass in kilodaltons. Bars, 10 µm.

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