Supplemental Figure 1.

1
Supplemental Figure 1.
A.
B.
2
Supplemental Figure 2.
Wildtype
K169R
R378A
vector
?118
none
RT-PCR products
3
Supplemental Figure 1. Characterization of
affinity purified Aur-A antibodies. A. Western
blots Protein samples taken from reticulocyte
lysate, reticulocyte lysate expressing Aur-A, and
oocyte extracts were analyzed by SDS-PAGE
followed by western blotting with the indicated
antibodies. The equivalent of one µl of oocyte
extract was loaded per lane, and the equivalent
of 5 µl of in vitro translation product or
reticulocyte lysate was loaded per lane. B.
Immunoprecipitation 30 ul of the affinity
purified Aur-A antibodies and 100 ul Dynabeads
were incubated in Buffer 1 (0.1M sodium phosphate
buffer, pH 8.1) for 1 hour at 4C. Samples were
washed twice in Buffer 1 and once in CSF-XB (see
methods). The beads were then added to 25 µl of
CSF extract. To recover Aur-A, two rounds of
precipitation were carried out. The equivalent
of 1 µl of extract was loaded per lane. More
than 80 of Aur-A was removed by
immunodepletion. Supplemental Figure 2.
Cell lines infected with Aur-A constructs
express Aur-A mRNAs. RNA was isolated from
NIH3T3 cells expressing the indicated constructs.
The equivalent of 1 µg of RNA was used per
RT-PCR reaction (Invitrogen SuperScript One-Step
RT-PCR). Reaction products were separated by gel
electrophoresis on a 1 agarose gel and detected
by ethidium bromide staining.