FIGURE 3.
Plg-RKT redirects GFP to the cell membrane. PC12 cells were transiently transfected with pAcGFP-Plg-RKT (in which the Plg-RKT cDNA was inserted in-frame for expression of a GFP-Plg-RKT fusion protein, with Plg-RKT at the C terminus) or with control pAcGFP vector. A, cell lysates were prepared and Western blotted with polyclonal anti-Plg-RKT IgG (lanes 1 and 2) or anti-GFP IgG (lanes 3 and 4). No reactivity was observed with preimmune IgG (not shown). B, the transfected cells were grown on coverslips and fixed in 2% formaldehyde and then washed and stained with a combination of DAPI and WGA-rhodamine at 20 °C in PBS. Cells were washed and mounted in IMMUNO-FLUORE mounting medium. Images were captured using a Zeiss confocal laser scanning microscope and then imported into the LSM Examiner and ImageJ programs for further processing as described under “Experimental Procedures.” The first and third panels of the images represent maximum projections of a series of optical slices through the cells. The GFP-Plg-RKT signal was localized primarily to the plasma membrane. The second and fourth panels of the images represent sagittal (apical-basal) slices through the same cells along the white dotted line indicated. Here, the peripheral plasma membrane localization of GFP-Plg-RKT is also evident throughout the vertical stacks of images that were acquired. The scale bar in the bottom right corner of the image represents 10 μm.