FIGURE 8.

TGase is constitutively active and localized to the leading edges of HeLa cells expressing an activated form of Ras and in MDAMB231 breast cancer cells. Serum-starved HeLa cells stably expressing the control vector (vector) or an activated form of Ras (Ras (G12V)), as well as parental HeLa and MDAMB231 cells were treated without (0) or with EGF for a day and then were lysed. A, extracts of the HeLa stables were immunoblotted with TGase, actin, and HA antibodies (top, second from the top, and third from the top panels, respectively). The same cell extracts were then assayed for transamidation activity as determined by the incorporation of BPA into lysate proteins (bottom panel). B, the parental HeLa and MDAMB231 cell extracts were also immunoblotted with TGase and actin antibodies (top and middle panels, respectively) and assayed for transamidation activity (bottom panel). C, stable HeLa cell lines and parental MDAMB231 cells were incubated with serum-free medium (Untreated), or the indicated combinations of serum-free medium ± EGF and ± MDC for a day and then were fixed. Immunofluorescence was performed on the samples using a TGase antibody and rhodamine-conjugated phalloidin (Actin). The resulting fluorescent images of the cells are shown. Membrane-localized TGase is indicated with arrows. D, the percentage of serum-starved HeLa cells expressing the control vector or Ras (G12V), or the parental MDAMB231 cells showing membrane-localized TGase was determined. Vector-only-expressing HeLa cells stimulated with EGF were used as a positive control. The results of three independent experiments were averaged together and graphed. The error bars indicate ±S.D.