Figure 7. Auxilin depletion causes... | Rockefeller University Press

Figure 7.

$Figure 7. Auxilin depletion causes sequestration of mitotic spindle proteins in clathrin cages. (A) Representative SILAC analysis of CCV fractions from control and auxilin-depleted cells, as performed in Fig. 1 E. The ranks of TACC3, GTSE1, and CKAP5 are indicated. (B) Western blot of whole cell lysates and CCV fractions corresponding to A. TACC3 and CKAP5 are barely detectable in control CCV fractions but highly enriched in CCV fractions from auxilin-depleted cells. In contrast, typical CCV proteins such as SNX9 are readily detectable in control CCVs. MHC-I is shown as a loading control. (C) Fractionation behavior of CKAP5, TACC3, and GTSE1 in control (left) and auxilin-depleted cells (right). In both cases, fractions were prepared using the original or the improved protocol, and compared by SILAC as in Fig. 1 F. CKAP5, GTSE1, and TACC3 (which was undetectable under control conditions) fractionate like clathrin-associated proteins only in auxilin-depleted cells, which indicates a specific association with clathrin cages. (D) Auxilin depletion causes loss of TACC3 from the mitotic spindle in HeLa cells. Bars, 10 µm. (E) HeLa cells were depleted of auxilin, clathrin, or both, or treated with a nontargeting siRNA (control), then stained with Hoechst to visualize DNA. Micronuclei (arrowheads) were detected by automated microscopy. Representative images are shown. Bar, 20 µm. (F) Relative changes in the proportion of micronucleated cells, normalized to nontargeting siRNA-treated cells (control). Error bars show the SEM of four independent repeats (three repeats for untreated cells). (G) Binary comparisons of the treatment groups shown in F, using ANOVA and the post-hoc Tukey-Kramer significance test. The difference between auxilin depletion and combined clathrin/auxilin depletion is highly significant (P < 0.001). The total number of cells scored for every condition is indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Corresponding Western blot analysis of cell lysates. Clathrin and auxilin (isoforms 1 and 2) were effectively depleted in the individual and the combined knockdowns. Positions of protein molecular mass markers are indicated in kilodaltons in B and H. In some cases, the apparent protein molecular weight is shown instead (indicated by ∼); this was estimated from the average migration of the protein relative to molecular weight markers on at least three gels.$

Auxilin depletion causes sequestration of mitotic spindle proteins in clathrin cages. (A) Representative SILAC analysis of CCV fractions from control and auxilin-depleted cells, as performed in Fig. 1 E. The ranks of TACC3, GTSE1, and CKAP5 are indicated. (B) Western blot of whole cell lysates and CCV fractions corresponding to A. TACC3 and CKAP5 are barely detectable in control CCV fractions but highly enriched in CCV fractions from auxilin-depleted cells. In contrast, typical CCV proteins such as SNX9 are readily detectable in control CCVs. MHC-I is shown as a loading control. (C) Fractionation behavior of CKAP5, TACC3, and GTSE1 in control (left) and auxilin-depleted cells (right). In both cases, fractions were prepared using the original or the improved protocol, and compared by SILAC as in Fig. 1 F. CKAP5, GTSE1, and TACC3 (which was undetectable under control conditions) fractionate like clathrin-associated proteins only in auxilin-depleted cells, which indicates a specific association with clathrin cages. (D) Auxilin depletion causes loss of TACC3 from the mitotic spindle in HeLa cells. Bars, 10 µm. (E) HeLa cells were depleted of auxilin, clathrin, or both, or treated with a nontargeting siRNA (control), then stained with Hoechst to visualize DNA. Micronuclei (arrowheads) were detected by automated microscopy. Representative images are shown. Bar, 20 µm. (F) Relative changes in the proportion of micronucleated cells, normalized to nontargeting siRNA-treated cells (control). Error bars show the SEM of four independent repeats (three repeats for untreated cells). (G) Binary comparisons of the treatment groups shown in F, using ANOVA and the post-hoc Tukey-Kramer significance test. The difference between auxilin depletion and combined clathrin/auxilin depletion is highly significant (P < 0.001). The total number of cells scored for every condition is indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Corresponding Western blot analysis of cell lysates. Clathrin and auxilin (isoforms 1 and 2) were effectively depleted in the individual and the combined knockdowns. Positions of protein molecular mass markers are indicated in kilodaltons in B and H. In some cases, the apparent protein molecular weight is shown instead (indicated by ∼); this was estimated from the average migration of the protein relative to molecular weight markers on at least three gels.