Figure 1.

Establishment of wild-type Rap1b-expressing Swiss 3T3 cell lines. (A) Expression of HA-tagged Rap1b as evaluated by immunoprecipitation-coupled blotting techniques by using a monoclonal anti-HA antibody (HA.11, Babco, Richmond, CA). Samples were analyzed on nonreducing SDS/PAGE with lysates from control (pL7-Hy) and two independent Rap1b-expressing clones (pL7-3-24 and pL7-3-50). HC and IgG indicate the position of heavy chain and whole Ig, respectively. (B) pL7-3-50 cells were analyzed by indirect immunofluorescence with the HA.11 antibody (dilution, 1/150). HA-tagged Rap1b shows a perinuclear localization, as reported for endogenous Rap proteins. (C) Morphological changes induced by wild-type Rap1b. Cells plated on six-well dishes were photographed under low magnification 1 day after reaching confluency. Typical fields with foci-like structures are shown here for Rap1b-expressing cells, as compared with a single monolayer observed in control cells.