FIG. 8.

Transformation by proto-Lbc- and onco-Lbc-derived mutants measured by NIH 3T3 focus formation assay. (A) Transformation by proto-Lbc and its derived mutants. An equimolar amount of each plasmid was used based on 40 ng/dish of ONC 4A, except for PS-1 where a fivefold increase of plasmid was used. (C) Transformation by onco-Lbc and derived mutants. Equimolar amounts of ONC 4A and TR4 were used; for the remaining DH and PH domain mutants, a fivefold increased amount of plasmid was used. Results are derived from triplicate transfections in a representative experiment and represent the means and SD of NIH 3T3 focus numbers generated. Similar results were obtained in at least two other independent experiments. (B and D) Western blot analysis of expression of the Lbc mutants in NIH 3T3 foci. Individual NIH 3T3 foci were ring cloned and expanded for cell lysis. Cell lysates (equal protein content) were immunoblotted with anti-Flag M2 antibody. In the case of PS-1, YDH, and NODH 4 mutants, which do not generate foci, the same plasmid amounts used for the focus formation assay were used to generate G418-selected NIH 3T3 transfectant colonies; individual colonies were ring cloned and expanded for cell lysis, and lysates were immunoblotted as described above.