Anti gata2

Cells were lysed using RIPA lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% Igepal, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche, Nutley, NJ). Whole-cell lysates (20 μg), whose protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, Fremont, CA), were run on 8% SDS Polyacrylamide gel for Western blotting. The following antibodies were used: anti-GATA2 (Novus Biological), anti-c-Myc (Cat No Sc-40, Santa Cruz Biotechnology, Santa Cruz, CA), anti-UHRF1 (Cat No MABE308, Millipore, Billerica, MA), anti-BIRC5 (Cat No 2808, Cell Signaling Technology, Danvers, MA) and anti-β-actin (Cat No L002, Epitope Biotech Inc., Burnaby, BC, Canada).

Serum-starved (overnight) cells on cover slips were incubated with nocodazole (10 μM; Sigma) for 4 hours [38 ]. The cells were then washed with serum-free medium (3x) to remove the drug and the cover slips collected at various time intervals. Cells were fixed with 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.5% Triton-X100 in PBS for 10 min. For immunofluorescence staining, cells were stained with anti-vinculin (Cat No V4505, Sigma), and anti-GATA2 (Novus Biological); secondary antibodies were obtained from Jackson Immuno Research (West Grove, PA). Slides were mounted using DAPI mounting solution (Vector Laboratories, Burlingame, CA) and viewed using a LSM 780 Confocal Microscope (Carl Zeiss Inc.).