FIG. 4.

Identification of the region within the 18E4 protein that is necessary for G₂/M arrest. (A) Schematic representation of the mutant 18E4 proteins tested. The truncated regions of the FLAG-tagged 18E4 protein are represented by black-gray bars. The motifs and functional regions reported for 16E4 are indicated (45). The corresponding regions in 18E4 are represented by gray boxes, and the amino acid sequence alignment of the conserved regions is presented. (B) Steady-state levels of mutant 18E4 proteins expressed in transfected cells. Both detergent-soluble (s) and non-detergent-soluble (i) fractions were analyzed by SDS-PAGE, followed by immunoblot analysis with an anti-FLAG antibody. The two panels represent short (upper) and long (lower) exposures of the same film. The positions of molecular weight markers (in thousands) are indicated on the left. (C) Cell cycle profiles of HeLa cells expressing the indicated 18E4 mutants. Cells were transfected with 4 μg of an E4 expression plasmid and pGreenLantern-1. FACScan analysis was performed as described in the legend to Fig. 2A. Cr, control; WT, wild type.