Fig. 2. Biochemical detection of SgP-CgA-GFP in sucrose density gradient fractions and in the secreted medium from PC12 cells. (A) Subcellular localization of SgP-CgA-GFP in PC12 cells. Postnuclear supernatants prepared from [³H]-L-norepinephrine-labeled PC12 cells transiently expressing SgP-CgA-GFP were centrifuged to equilibrium on sucrose density gradients. Fractions were assayed for sucrose concentration, GFP fluorescence and scintillation counting. (B) Immunochemical detection of the SgP-CgA-GFP chimera in sucrose density gradient fractions. Gradient fractions were concentrated using SepPak C-18 cartridges and processed for immunoblot using an anti-GFP antibody. (C) Immunochemical detection of stimulated release of SgP-CgA-GFP or proANF-GFP. Regulated secretion from SgP-CgA-GFP- or proANF-GFP-expressing PC12 cells was triggered by 2 mM BaCl₂. Extracellular media were concentrated using C-18 SepPak cartridges and processed for immunoblot using an anti-GFP antibody. Immunoreactivity was visualized by chemiluminescence.