FIG 1.

ECD localization and expression do not change during cell cycle progression. 76NTERT cells were cell cycle arrested by culturing in growth factor-free DFCI-3 medium for 72 h and then switched to growth factor-containing DFCI-1 medium to initiate cell cycle progression. (A) Cells were fixed in 70% ethanol at the indicated time points, stained with propidium iodide, and subjected to FACS analysis. (B) Lysates were collected at the indicated time points and subjected to Western blotting for ECD or β-actin. ImageJ software was used to quantify ECD signals at various time points during cell cycle progression and expressed as relative to β-actin signals. (C) Nuclear (N) and cytoplasmic (C) fractions were isolated from cells at various time points during cell cycle progression and subjected to Western blot analysis with anti-ECD antibody. PARP and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) served as positive controls for nuclear and cytoplasmic proteins, respectively. All experiments were carried out in triplicate. H.E., higher exposure.