FIG. 1.

Involvement of amino-terminal Zta sequences in growth arrest and induction of p53, p21, and p27. (A) Schematic representation of Zta structure. (B and C) Cell cycle analysis (B) and Western blot analysis of the cell cycle regulatory proteins p53, p21, p27, and pRb (C) following induction of the indicated Zta proteins in stably transfected HeLa cells. The indicated cell lines were generated simultaneously, and induction experiments were performed in parallel. Cells were expanded in the presence of tetracycline (2 μg/ml). Cells were then trypsinized, washed twice with 1× PBS, and either collected (for 0-h time point) or plated in medium with no tetracycline for the indicated times. A portion of collected cells were stained with propidium iodide and subjected to FACS analysis to determine the DNA content (B). (C) The remaining cells were suspended in Laemmli buffer and boiled for 20 min to reduce the viscosity of the samples. A portion of these samples were subjected to SDS-PAGE, transferred to nitrocellulose, and subjected to sequential Western blot analysis (with a stripping step between probings) using anti-Zta (M47), anti-p53 (DO-1; Santa Cruz), anti-p21 (C19G; Santa Cruz), anti-Kip1/p27 (Transduction Laboratories), and anti-pRb (G3-245; PharMingen) antibodies. wt, wild type.