Modulation of α-tubulin detyrosination levels by manipulation of vasohibins- SVBP impacts chromosome segregation fidelity. (A, C, and D) Immunoblot analysis of detyrosinated α-tubulin and total α-tubulin levels after VASH1-SVBP overexpression, RNAi mediated knockdown of TTL or VASH1 and VASH2 (with/without SVBP), respectively. GAPDH was used as loading control. The expression of VASH1-GFP and SVBP-myc was determined using anti-GFP and anti-myc antibodies, respectively. (B and E) Representative scanning confocal microscopic images of metaphase spindles immunostained with antibodies against detyrosinated and tyrosinated α-tubulin for each condition. DNA was counterstained with DAPI. Scale bars, 10 µm. (F) Representative images from spinning-disk confocal time-series displaying different stages of mitosis in U2OS cells stably expressing H2B-GFP (cyan)/mCherry-α-tubulin (red) transfected with the indicated plasmids and siRNAs. White arrowhead points to a segregation error during anaphase. Scale bars, 10 µm. Time in hours:minutes. (G) Quantification of the percentage of anaphase cells with segregation defects (lagging chromosomes and DNA bridges not discriminated) from spinning-disk confocal imaging of U2OS cells stably expressing H2B-GFP/mCherry-α-tubulin transfected with the indicated plasmids and siRNAs. Error bars indicate SD of the mean. N (number of cells, number of independent experiments): control(GFP)(38, 4); VASH1-GFP+SVBP-myc (38, 4); siTTL (28, 2); siControl (121, 5); siVASH1/2 (91, 4); and siVASH1/2+siSVBP (45, 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA.