Figure 6.

Nonphosphorylatable Ala-substituted Op18 deletion derivatives can be used as probes to distinguish interphase and mitotic MTs. K562 cells were transfected, and hygromycin resistant cell lines were selected as in Figure 1 and induced with Cd ²⁺ (0.5 μM) for the indicated time. To obtain comparable expression levels of the indicated Flag-epitope tagged Op18 derivatives, DNA concentrations were adjusted as indicated in MATERIALS AND METHODS. (A) Transfected cell lines were Cd²⁺ induced for 24 h, and phosphoisomers of Flag-epitope tagged Op18 were resolved by native PAGE and revealed using antibodies toward the epitope tag. The expressed derivatives migrate at similar positions, as the corresponding E. coli produced derivatives (data not shown). (B) Op18 levels were determined after 6 and 24 h of Cd²⁺ induction, and data are presented as fold induction over endogenous Op18 (∼ 0 μM). (C) The fraction of polymerized tubulin in the cells shown in (B). Panels B and C show the mean of two independent transfection experiments.