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. 2009 May 7;42(2):181–189. doi: 10.1165/rcmb.2009-0058OC

Figure 2.

Figure 2.

Intracellular trafficking and processing of WT proSP-C in human embryonic kidney (HEK) 293 cells. (A) HEK293 cells were transfected with a construct encoding human WT proSP-C (SP-C1–197) cloned into pIRES2-EGFP. After 24 hours, lysates from cells transfected with lipofectamine alone (lipo), pIRES2-EGFP vector, or plasmid encoding SP-C1–197 were analyzed by SDS-PAGE/Western blotting with antibody directed against the NH2-terminal propeptide of proSP-C (proSP-C); blots were subsequently stripped and reprobed with antibodies directed against the lumenal domain of SP-C (SP-Cc) or the mature SP-C peptide. Short arrow indicates proSP-C; open arrowhead indicates mature SP-C. (B) Cell lysates from HEK293 cells transfected with SP-C1–197 (transfected HEK) were mixed with human bronchoalveolar lavage fluid (hBALF) and analyzed by SDS-PAGE, followed by Western blotting with antibody directed against mature SP-C peptide (left panel). In a separate experiment (right panel), HEK293 cells transfected with plasmid encoding SP-C1–197 (transfected), untransfected HEK293 cells (untransfected), or hBALF were extracted with chloroform/methanol, and the organic phase analyzed by Western blotting with antibody directed against the mature SP-C peptide. Open arrowhead indicates mature SP-C peptide in both panels. (C) HEK293 cells were transfected with SP-C1–197 cloned into pIRES2-EGFP, and treated with brefeldin A (5 μg/ml) or vehicle control (MeOH) for 20 hours. Cell lysates were prepared 24 hours after transfection, and analyzed by SDS-PAGE/Western blotting. Blots were serially probed with antibody directed against the N-terminal peptide of proSP-C, SP-C mature peptide, or GFP. Long arrow indicates GFP (encoded by the bicistronic pIRES2-EGFP vector); short arrowhead indicates proSP-C; and open arrowhead indicates mature peptide. (D) HEK293 cells were transfected with SP-C1–197 cloned into pIRES2-EGFP. Cells were permeabilized 24 hours after transfection, stained with antibodies directed against Lamp-1 or mature SP-C peptide, and analyzed by confocal microscopy. Upper left panel is signal from EGFP (encoded by the pIRES2 vector), which is expressed in the cytosol and delineates the boundary of the cell.