Supplement Figure 1. Purification of GST-NDEL1 from stably transformed MEFs.
A Generation of a MEF line in which endogenous NDEL1 was replaced by GST-NDEL1
using NDEL1 disrupted mice (33). The distribution of GST-NDEL1 was
indistinguishable compared to endogenous NDEL1. B GST-NDEL1 was expressed at
relatively normal amounts compared to endogenous NDEL1, which was confirmed by
Western blot using an anti-NDEL1 antibody. C Purification of GST-NDEL1. Note that
only endogenous GST was isolated from untransfected MEFs. D Characterization of
phosphorylation of NDEL1 by LC/MS/MS. Phosphorylation of T219 and S231 was
detected, presumably due to the phosphorylation by CDK1. We also detected a novel
S251 phosphorylation event. E Demonstration of direct interaction between
NDEL1/mutated NDEL1s (S251A, S251E) and Aurora-A by immunoprecipitation.
Lysates of cells expressing either GFP-NDEL1/ mutated NDEL1s (S251A, S251E) and
Flag-tagged wild-type Aurora-A were subjected to immunoblot analysis with antibodies
to Flag or to GFP. The same cell lysates were also subjected to immunoprecipitation with
antibodies to GFP (left) or Flag (right), and the resulting precipitates were subjected to
immunoblot analysis with antibodies to Flag (left) or to GFP (right) as indicated.
Supplement Figure 2. Generation of a phosphorylated NDEL1 specific monoclonal
antibody.
Characterization of an anti-P-NDEL1 (phospho-Ser251-specific) monoclonal antibody. A
Anti-P-NDEL1 mAb specifically recognized phospho-NDEL1 proteins that were
phosphorylated with Aurora-A in vitro. Incorporation of phosphate was examined by 32P.
A specific signal was detected from in vitro phosphorylated NDEL1 by the anti-phospho-
S251 monoclonal antibody. B Western blot analysis revealed that the anti-P-NDEL1
antibody specifically recognized phospho-Ser251 of NDEL1. Anti-P-NDEL1 antibody
was pre-absorbed with phospho-peptide S251S-P of various concentrations and then was
used for Western blotting. The phospho-peptide S251S-P efficiently and specifically
suppressed the signal detected by the anti-P-NDEL1 antibody. C Examination of an anti-
P-NDEL1 antibody using GST-NDEL1 extracted from synchronized HeLa cells which
were transfected with a GST-NDEL1 expression vector. The anti-phospho-S251 antibody
specifically recognized GST-NDEL1 which was extracted from G2-prophase.
Phosphatase treatment of the protein extract significantly reduced the signal. In addition,
there was no obvious signal in the extract from HeLa cells which were transfected by
GST-NDEL1(S251A). D Cre mediated Ndel1 disruption in MEF cells reduced the signal
which was detected by the anti-phospho-S251 antibody as well as the anti-NDEL1
antibody. E The signal detected by the anti-phospho-S251 monoclonal antibody was
reduced in protein extracts from HeLa cells treated by siRNA against Aurora-A. F
Immunohistochemistry revealed that the signal detected by the anti-phospho-S251
monoclonal antibody was not detected in HeLa cells which were treated by siRNA
against Aurora-A.
Supplement Figure 3. Confirmation of the expression GFP-NDEL1 and mutated
GFP-NDEL1s in MEF cells.
Western blot using an anti-GFP and the anti-NDEL1 antibodies. Synchronized MEF cells
were transfected by expression vectors carrying wild type GFP-NDEL1, and mutated
GFP-NDEL1s (indicated above panel). Endogenous Ndel1 signal disappeared after
replacement.
Supplement Figure 4. Biacore sensorgrams and quantitation of protein interaction.
A Characterization of binding of the recombinant protein of GST-NDEL1, GST-TACC3
was performed using surface plasmon resonance detection. GST- TACC3 was
immobilized on the sensor chip surface. The traces are representative of three different
experiments. The specific binding signal shown was obtained by subtracting the signal
measured in the absence of immobilized recombinant proteins from the signal in the
presence of the fusion protein. The relative change in refractive index expressed as a
change in resonance angle (RU) as binding of GST-NDEL1 to an immobilized GST-
TACC3 on a sensor surface in the BIAcore (BIAcore AB) apparatus was monitored over
time. The binding affinity between NDEL1 and TACC3 is extremely strong as indicated
(KA(1/M): 9.45x109±3.52x109). B We performed BIAcore analysis using the opposite
combinations of proteins used in above. GST-NDEL1 was immobilized on the sensor
chip surface. The similar strong binding affinity between these two proteins was observed
(KA(1/M): 1.33x1010±0.34x1010). C We examined whether mutation of NDEL1 at
phosphorylation site influences on the binding affinity with TACC3. We immobilized
GST- TACC3 on the sensor chip surface, and loaded mutated NDEL1s. Either alanine
mutants or glutamic acid mutant at S251 did not reveal significant change of binding
affinity (KA(1/M)(S251A): 9.82x109±3.92x109: KA(1/M)(S251E): 8.98x109±4.01x109),
suggesting that phosphorylation of NDEL1 by Aurora-A is essential for centrosomal
targeting rather than binding to other protein including TACC3.