Fig. 1

Phosphorylation of TACC3 at Ser 558 is involved in astral microtubule assembly at the centrosomes. a. Schematic representation of GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (S558A)-TACC3 shRNA and the GFP-TACC3 (S558D)-TACC3 shRNA constructs. b. Lysates of HeLa cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (S558A)-TACC3 shRNA and the GFP-TACC3 (S558D)-TACC3 shRNA for 48 h were analyzed by Western blot to detect the levels of exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and the exogenous TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. Actin was probed as control. c. Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (S558A)-TACC3 shRNA or GFP-TACC3 (S558D)-TACC3 shRNA transfected mitotic HeLa Kyoto cells showing the differences in astral microtubules. Scale bar, 5 μm, Microtubules (red channel shown in grey) were stained with mouse monoclonal anti-α-tubulin antibody. The arrows show centrosomes/spindle poles. Centrosomal regions are also shown in enlarged view. d. Bar graph shows the quantification of metaphase cells with astral microtubule assembly defects in different conditions as of C in HeLa Kyoto cells. Percentage refers to the ratio of the number of metaphase cells with loss of asters and the total number of metaphase cells. The bars represent mean +/− S.E. Number of mitotic cells counted = 50 each (three independent experiments)