Fig. 4. Unprocessed EGFP/hSP-C^Exon4 accumulates in juxtanuclear inclusions. (A-D) Immunofluorescence of A549 cells grown on coverslips were transfected with either EGFP/hSP-C^1-197 (A) or EGFP/hSP-C^Exon4 (C). Forty-eight hours after introduction of plasmid DNA, cells were fixed and fluorescence images acquired. Corresponding phase images are presented in panels B and D; (E) Western blotting for detection of EGFP proteins. Nuclear-free lysates were prepared as detailed in Materials and Methods using cell pellets from dishes transfected identically to those in panels A-D. Fifty percent of each lysate was subjected to 12% SDS-PAGE. Separated proteins were transferred to nitrocellulose and immunoblotted with primary rabbit polyclonal anti-GFP. Bands were visualized using enhanced chemiluminescence. EGFPC₁ was expressed as a major product with M_r 27,000 (lane 1). Analysis of EGFP/hSP-C^1-197 fusion protein expression (lane 2) demonstrated two doublets bands with relative molecular weight (M_r) of 48-50,000 and 33-38,000, consistent with a primary translation product/palmitoylated form and two processing intermediates. In contrast, EGFP/hSP-C^Exon4 was expressed as a single smaller band (hatched arrow, lane 3).