Figure 4. Analysis of amelotin secretion from C2C12 cells by immunocytochemistry and Western blot. Expression of the FLAG peptide alone (cFLAG-pCMV5) shows a strong signal in the cytoplasm; Western analyses of cell lysates (a) and conditioned media (b) show no signal, since the FLAG peptide is too small to be resolved by PAGE. Expression of the amelotin-FLAG fusion protein (cFLAG-Amelotin) shows a weak signal in the cytoplasm and a strong signal in the conditioned media, while the addition of Brefeldin A (cFLAG-Amelotin + Brefeldin A) causes retention of the amelotin fusion protein in the cytosol. Bar indicates 50µm.