with IP buffer B (150 mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, 0.1% Triton X-100, pH 7.4). After the final wash, the supernatant was removed and 50 ml of 46 SDS sample buffer were added. Samples were boiled for 5 minutes at 100uC, spun down, and separated by SDS-polyacrylamide gels for Western blotting. In vitro kinase assay and mass spectrometry For each reaction, 200 units of recombinant Aurora A (Cell Biosciences; Palo Alto, CA) were incubated with 1 mg recombinant centrin-2 for 30 minutes at 30uC in the presence or absence of 10 mM ATP in kinase buffer (50 mM Tris, 10 mM MgCl2, 0.16 mg/ml DTT, pH 7.4). After the initial 30 minutes, another 10 mM ATP, or equivalent volume of kinase buffer minus ATP, was added and the mixture was allowed to incubate for an additional 30 minutes at 30uC. The reaction was stopped by adding SDS buffer to the samples and boiling the samples. The kinase reactions plus and minus ATP were separated by SDS- PAGE, Coomassie stained, and sent for mass spectrometry analysis to determine phosphorylated residues in centrin. Immunofluorescence and microscopy Cells were grown on coverslips, washed once with MTSB (3 mM EGTA, 50 mM PIPES, 1 mM magnesium sulfate, 25 mM potassium chloride), and fixed in 220uC methanol for 10 minutes. Coverslips were dried at room temperature, rehydrated in 0.05% triton X-100/PBS for 3 minutes, and blocked in goat blocking buffer (5% goat serum, 1% glycerol, 0.1% BSA, 0.1% fish skin gelatin, 0.04% sodium azide, pH 7.2) for at least 30 minutes. Coverslips were incubated in primary antibody for 1 hour at room temperature, washed three times with PBS, incubated in secondary antibody for 1 hour at room temperature, and washed again as above. Coverslips were post-fixed in 4% EM grade paraformalde- hyde (Electron Microscopy Sciences, Hatfield, PA) in PBS for five minutes, washed twice, dried, and mounted onto slides with ProLong Gold reagent with DAPI (Invitrogen, Carlsbad, CA). Tissues were treated as described above but were freshly cryosectioned from frozen breast tissue the day of the staining and the MTSB wash step was omitted. Images were collected as Z-stacks on a LSM 510 Confocal Laser Scanning Microscope (Zeiss, Germany) for all images except those in Figure 4A+B, which were collected on a Zeiss Axioskop. To improve their quality, we performed minimal image adjustments on them using PhotoShop as follows. Using ‘‘Levels’’, both images had their RGB input settings changed to 10, 0.79, and 255. The inset images had blue removed. The inset images then had the green levels adjusted to minimize the green ‘‘haze’’ from the GFP transfection marker so that the green AlexaFluor 488 signal was clearer. No other post-capture image processing was performed. Stable HeLa Tet-On centrin mutant cell lines The HeLa Tet-On system was purchased from Clontech. Mutations in the centrin-2 sequence, cloned into the pTRE2hygro- HA vector, were created using site-directed mutagenesis. The constructs were transfected into HeLa Tet-On cells containing the pTRE-Tight vector using FuGene 6 (Roche) according to manufac- turer’s instructions. Stably transfected cells were selected by exposing cells to hygromycin and screened using doxycycline induction. Pulse/wash-out experiments and proteasome inhibition For pulse/wash-out experiments, HeLa Tet-On cells were treated with doxycycline for 12 hours, washed with DMEM medium, then incubated in normal Tet-On growth medium lacking doxycycline. Samples were harvested at indicated times and lysed in Control of Centrin Stability by Aurora A SDS lysis buffer as described above. For proteasome inhibition studies, cells were incubated with DMSO as a vehicle control, 100 mM ALLnL or MG132, 20 mM ammonium chloride, or 10 mM leupeptin for at least six hours. Cells were then harvested at indicated times and lysed in SDS lysis buffer containing N- ethylmaleimide (NEM) at a final concentration of 1 mg/ml. Expression vector and shRNA construction and knock down of Aurora A and centrin Aurora A cDNA (NCBI accession # NM_003600.2) was purchased from Origene. It was amplified by standard PCR and cloned into pEGFP-C3 vector (Clontech). shRNA oligos directed against the 39 UTR of Aurora A (TAGGGATTTGCTTGG- GATA) were annealed and cloned into an RNA polymerase III dependent H1 RNA promoter driven vector. The same process was repeated for the shRNA directed against the 39UTR of centrin-2 (AGCTTTGAGCACCTGCCATTT). HeLa and HeLa Tet-On cells were transfected with the Aurora A expression vectors and/or Aurora A or centrin-2 shRNA using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Doxycycline was added to the cells 4 hours post-transfection and cells were harvested at the indicated times and counted for cell proliferation assays or lysed for Western blot analysis. Statistical analysis Centrin mutant half-life was determined by plotting the optical density of Western blot analyses normalized against beta-actin optical density for each mutant at each time point in 5 independent experiments. A linear trendline was plotted for each curve, and the half-life was determined from the slope of the trendline. The resultant half-life values for each mutant were averaged and the standard deviation and standard error were calculated. The probability that variances between the mutants were not significantly different was determined using an F-test. For the cell proliferation assay probability, a paired Student’s T Test was performed on the data normalized against the wildtype results. Supporting Information Figure S1 DNA content of synchronized HeLa cells. HeLa cells harvested at the indicated time points after synchronization by double thymidine/nocodazole block and release. Cells were fixed with ethanol and stained with propidium iodide to measure DNA content. (DOC) Figure S2 Aurora A knock down in HeLa cells. HeLa cells were transfected with Aurora A shRNA and whole cell lysates were harvested 24 hours after transfection. Whole cell lysates were separated by SDS-PAGE and blotted with the indicated antibodies. (DOC) Figure S3 Centrin knock down in HeLa Tet-On HA-Centrin expressing cells. HeLa cells were transfected with centrin shRNA and whole cell lysates were harvested 48 hours after transfection. Whole cell lysates were separated by SDS-PAGE and blotted with the indicated antibodies. Note that it was possible to induce the expression of HA-centrin (HA) while knocking down endogenous centrin (E). (DOC) Figure S4 Asynchronously cycling HeLa cells were fixed, permeabilized, and processed for immunofluorescence as de- scribed in the Material and Methods section. This image shows an interphase HeLa cell exhibiting faint p-S170 centrin (red, denoted with white arrowhead) co-localizing with Aurora A (green) staining at the centrosome. (DOC) PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e21291