Subcellular localization study of overexpressed hCoV-NL63 ORF 3 protein in Huh-7 cells. Confocal laser scanning microscopy on cells expressing recombinant ORF 3 protein and co-staining with different antibodies for cellular organelles. Left panels: staining with rabbit-anti-ORF 3 serum and anti-rabbit-Cy2 (Dianova). Middle panels from top to bottom: co-staining of cellular organelles with a mouse-anti-ERGIC53 (A), mouse-anti-Golgi 58 K for the Golgi (B), goat-anti-LAMP-1 for trans-Golgi Network (TGN) and Lysosomes (LYS) together with goat (or donkey)-anti-mouse-Cy3 antibodies (C). Right panels show merged pictures where yellow areas represent colocalization. Partial colocalizations can be observed with all organelle markers indicating that the glycoprotein ORF 3 is processed trans- Golgi. Bars indicate 20 μm.