Anti aurora b

Cells were seeded onto poly-l-lysine-coated coverslips, fixed in ice-cold methanol or 2% formaldehyde, washed three times in PBS, permeabilized in 0.25% Triton X-100 in PBS for 10 min, blocked in 0.2% Triton X-100, 5% bovine serum albumin in PBS for 60 min before the required primary Abs were applied. The following Abs were employed: anti-Aurora-B (BD-Bioscience); anti-ECT2, anti-PRC1, anti-MKLP1, and anti-PLK1 (Santa Cruz); anti-HIPK2 (rabbit polyclonal Ab [19 (link)]); anti-p-histone-H2B-S14 (Cell Signaling); anti-p-histone-H2B-S32, anti-H2B, anti-53BP1, and anti-MgcRacGAP1 (Abcam); anti-β-tubulin-Cy3 and anti-α-tubulin-FITC (Sigma-Aldrich); secondary 488- or 594-conjugated Abs (Life Technologies). DNA was marked with Hoechst 33342 (Sigma-Aldrich) or Red-Dot2 far-red (Biotium). Cells were examined under an Olympus BX53 microscope using a cooled camera device (ProgRes MF) and with confocal microscope Zeiss LSM510-Meta.

KIF5B antibody is homemade and has been described elsewhere (WB: 1:2000; immunofluorescences: 1:250) [5 (link)]. Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLight™ Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes). For Western Blot secondary antibodies were from Zymed^® Laboratories. For immunofluorescence, the secondary antibodies conjugated to Alexa 488, 555, 649 were from Jackson ImmunoResearch Laboratories or Molecular probes (Additional file 8: Movie S1).

Staining was performed as described^{28 (link),55 (link),56 (link)}. Primary antibodies: mouse monoclonal anti-mCherry (1:200, Clontech, 632543 or 1:50, DSHB, 3A11), anti-Tropomyosin (1:200, Sigma, T9283), anti-sarcomeric alpha Actinin (1:100, Abcam, ab9465), anti-Aurora B (1:200, BD Transduction Laboratories, 611083), anti-p27 (1:50, BD Transduction Laboratories, 610242), anti-Ki67 (1:250, Abcam, ab8191), rabbit polyclonal anti-Troponin I (sc-15368), anti-Cyclin A (sc-751), anti-cdc2 (sc-954), anti-Geminin (sc-13015) (all 1:50, Santa Cruz), anti-phospho-Histone H3 (Ser10) (1:200, Millipore, 06-570), anti-pRb807/811 (1:100, Cell Signaling, 9308), anti-mAG (1:300, MBL, PM011), anti p27 (1:50, SantaCruz, sc-528), anti-survivin (1:50, Novus, NB500-201K8), rat monoclonal anti-BrdU (1:100, Abcam, ab6326) and goat polyclonal anti c-kit (1:100, R&D Systems, AF1356). Immune complexes were detected with ALEXA 488- or ALEXA 594-conjugated secondary antibodies (1:200; Molecular Probes). DNA was visualized with DAPI (4′, 6′-diamidino-2-phenylindole, 0.5 g/ml). For BrdU, cells were cultured in 30 μM BrdU (neonatal: last 24 h, adult: last 5 days).

Staining was performed as described (24 (link)). Primary antibodies: anti-tropomyosin (1:200, Sigma), anti-actinin (1:100, Abcam), anti-aurora B (1:200) (both BD Transduction Laboratories), rabbit polyclonal anti-troponin I, anti-cyclin A, anti-cyclin dependent kinase 1 (cdc2), anti-geminin (all 1:50, Santa Cruz Biotechnology), anti-phospho-histone H3 (Ser10) (1:200, Millipore), anti-pRb807/811 (1:100, Cell Signaling), anti-mAG (1:300, MBL), rat monoclonal anti-5-Bromo-2-deoxyuridine (BrdU) (1:100, Abcam). Immune complexes were detected with ALEXA 488- or ALEXA 594-conjugated secondary antibodies (1:200; Molecular Probes). DNA was visualized with DAPI'(4” 6'-diamidino-2-phenylindole, 0.5 μg/ml). For BrdU, cells were cultured in 30 μM BrdU (Sigma) (neonatal: last 24–48 h, adult: last 5 days).