Fig. 1. Characterization of transfected cells. REF-52 cells were transfected with -SMA-EGFP and cells stably expressing -SMA-EGFP were obtained after FACS sorting and G418 selection. (A) In the majority of cells, -SMA-EGFP is incorporated into actin stress fibers (see inset), as indicated by indirect double immunofluorescence of fixed cells. (a) -SMA was visualized with anti--sm1 antibody, (b) -SMA-EGFP was visualized with anti-GFP antibody. (c) Overlay of a and b. Scale bar, 50 µm. (B) Transfected REF-52 cells were grown for 5 days in culture medium containing either 2% FCS (a) or 20% FCS (b). Cells were fixed and -SMA-EGFP organization was assessed by fluorescence microscopy. Scale bar, 20 µm. (C) Cell homogenates of non-transfected cells (lanes 1 and 3) and transfected cells (lanes 2 and 4) were analyzed on a 10% SDS-PAGE and immunoblotted with anti-GFP antibody (lanes 1-2) and anti-SMA antibody (lanes 3-4). The anti-GFP antibody detected a single 80 kDa band corresponding to the calculated molecular mass of the fusion protein (42 kDa actin+36 kDa EGFP). This band was also recognized by the anti--SMA antibody. Transfected and non-transfected cells both showed the endogenous -SMA band of 42 kDa.