FIG. 7.

Expression and intracellular localizations of the p235 N-terminal and the C-terminal regions in transfected COS-7 cells. COS-7 cells were transiently transfected with the EGFP–Ns1-286 and EGFP–C1684-2052 cDNAs either on a 35-mm-diameter dish (A) or on coverslips (B) as indicated in Materials and Methods. (A) Cell lysates were collected in RIPA buffer 24 h after transfection and subjected to SDS-PAGE, electrotransfer to nitrocellulose membranes, and immunoblotting with anti-GFP monoclonal antibody (2 μg/ml). (B) Cells were washed and fixed in methanol 15 h after transfection with the indicated constructs. Localization of the expressed proteins was determined by the fluorescence signals of GFP with a standard fluorescein filter of a Zeiss confocal microscope. Characteristic targeting of EGFP–Ns1-286 protein to vesicle structures reminiscent of endosomes is observed at both low (b) and high (c) levels of expression. No punctate peripheral staining is associated with EGFP–C1684-2052 (d), but rather perinuclear and diffuse green fluorescent signals, excluded from the nucleus, are observed.