Fig. 3. Modulation of MAP4-mediated MT stabilization by two distinct Op18 truncation derivatives in the G2-phase population. (A) Native Op18 is depicted with an N-terminal region (residues 1-45) and an extended -helical region that contain two homologous repeats separated by 51 residues (designated Repeat 1 and Repeat 2), each of which binds an /ß-tubulin heterodimer indicated by open and filled circles (Gigant et al., 2000). Each truncated Op18 derivative is denoted by the numbers within brackets, which indicate the amino acid residues present. As depicted in the figure, the N-terminally truncated Op18(25-149) derivative retains two-site positive binding cooperativity, which facilitates tubulin sequestering, whereas the Op18(1-99) derivative binds single heterodimers with low affinity but still promotes catastrophes via the intact N-terminus (Howell et al., 1999; Larsson et al., 1999). (B) Cotransfected K562 cell lines (DNA ratio 1:2 of MAP4/Vector-Co: truncated Op18/Vector-Co) were induced with Cd²⁺ for 20 hours. Immunoblots of cellular lysates were probed with anti-SLEEIQ, which recognizes full-length and the two truncated Op18 derivatives with similar efficiency. Mean MAP4-specific fluorescence intensities, as determined by flow cytometry, are given below the autoradiograph. Mean MT-specific fluorescence within the G2 populations was also determined after 20 hours of induced expression and is shown in the bottom panel. The original histogram data, from which the mean fluorescence intensities were derived, included >95% of all cells and revealed a well-defined single peak in all cases (data not shown). All data in this figure are derived from the same transfected cell populations but are representative of at least three independent transfection experiments.