Fig. 6. Mutation of Cys120/Cys121 in the COOH flanking propeptide mimics EGFP/hSP-C^Exon4 expression. A549 cells were transiently transfected with EGFP/hSP-C^1-197 (A,B) or the COOH cysteine mutant EGFP/hSP-C^C120/121G (C,D) using CaPO₄. Images for EGFP expression were acquired 48 hours after transfection by using fluorescence microscopy with a FITC filter package. As for Exon4, the cysteine mutant was restricted to a juxtanuclear region showing aggregation. (E) Western blotting for detection of EGFP proteins. Nuclear-free lysates were prepared from cell pellets obtained from dishes identically transfected with EGFP/hSP-C^C120/121G, EGFP/hSP-C^{C120/121/189G}, EGFP/hSP-C^C189G, EGFP/hSP-C^1-197, or EGFP/hSP-C^Exon4 as detailed in Materials and Methods. Immunoblotting was performed with primary rabbit polyclonal anti-GFP and bands visualized using enhanced chemiluminescence. EGFPC₁ was expressed as a major product with M_r 27,000 (not shown). Analysis of EGFP/hSP-C^1-197 fusion protein expression (lane 5) demonstrated a primary translation bands with relative molecular weight (M_r) of 50-48,000 and processed forms of 37-33,000. EGFP/hSP-C^C120/121G (lane 3), EGFP/hSP-C^{C120/121/189G} (lane 4), and EGFP/hSP-C^C189G (lane 1) were each expressed as a single band with M_r 48,000. Mock transfected cells (lane 2) yield no immunoreactive bands.