Figure 4.
NuSAP negatively regulates the concentration of Kif2A on spindle poles and promotes the localization of Eg5 on spindle microtubules. (A and E) Control and NuSAP-knockout HeLa cells were stained with anti-Kif2A (red) and anti-α-tubulin (green) antibodies in A and stained with anti-Eg5 (red) and anti-α-tubulin (green) antibodies in E. DNA was stained with DAPI (blue). (B and F) Quantification of relative Kif2A (B) and Eg5 (F) fluorescence intensity on the spindle in A and E, respectively. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P < 0.0001 in B; P = 0.0080 in F. (C and G) Mitotic HeLa cells were transfected with GFP or GFP-NuSAP (green) and stained with anti-Kif2A (red) and anti-α-tubulin (magenta) antibodies in C, and stained with anti-Eg5 (red) and anti-α-tubulin (magenta) in G. DNA was stained with DAPI (blue). (D and H) Quantification of relative Kif2A (D) and Eg5 (H) fluorescence intensity on the spindle in C and G, respectively. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0004 in D; P = 0.0014 in H. (I and J) Mitotic HeLa cells were subjected to an IP assay using nonspecific IgG or anti-NuSAP antibodies followed by Western blot analysis with anti-NuSAP, anti-Eg5 (I), and anti-Kif2A (J) antibodies. (K) Test the protein level of Kif2A and Eg5 after NuSAP knockdown in HeLa cells. (L) The mean velocity of poleward spindle microtubule flux in Kif2A-depleted and NuSAP/Kif2A-co-depleted cells. Error bars indicate SD. Three independent replicates of 13 or 14 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.9884. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n = the number of independent experiments presented. Scale bars, 10 μm. Source data are available for this figure: SourceData F4.

NuSAP negatively regulates the concentration of Kif2A on spindle poles and promotes the localization of Eg5 on spindle microtubules. (A and E) Control and NuSAP-knockout HeLa cells were stained with anti-Kif2A (red) and anti-α-tubulin (green) antibodies in A and stained with anti-Eg5 (red) and anti-α-tubulin (green) antibodies in E. DNA was stained with DAPI (blue). (B and F) Quantification of relative Kif2A (B) and Eg5 (F) fluorescence intensity on the spindle in A and E, respectively. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P < 0.0001 in B; P = 0.0080 in F. (C and G) Mitotic HeLa cells were transfected with GFP or GFP-NuSAP (green) and stained with anti-Kif2A (red) and anti-α-tubulin (magenta) antibodies in C, and stained with anti-Eg5 (red) and anti-α-tubulin (magenta) in G. DNA was stained with DAPI (blue). (D and H) Quantification of relative Kif2A (D) and Eg5 (H) fluorescence intensity on the spindle in C and G, respectively. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0004 in D; P = 0.0014 in H. (I and J) Mitotic HeLa cells were subjected to an IP assay using nonspecific IgG or anti-NuSAP antibodies followed by Western blot analysis with anti-NuSAP, anti-Eg5 (I), and anti-Kif2A (J) antibodies. (K) Test the protein level of Kif2A and Eg5 after NuSAP knockdown in HeLa cells. (L) The mean velocity of poleward spindle microtubule flux in Kif2A-depleted and NuSAP/Kif2A-co-depleted cells. Error bars indicate SD. Three independent replicates of 13 or 14 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.9884. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n = the number of independent experiments presented. Scale bars, 10 μm. Source data are available for this figure: SourceData F4.

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