PreimmuneMonoclonalanti-Aur-AAffinity Purified anti-Aur-A Blot: retic lysate translation product oocyte extract retic lysate translation product oocyte extract retic lysate translation productoocyte extract retic lysate translation product oocyte extract preimmune Aur-A Immunodepleting antibody: Blot: Aur-A min. Supplemental Figure 1. A. B.

Supplemental Figure 2. nonevectorWildtypeK169RR378A∆118 RT-PCR products

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.