Protein samples normalized to equal midgut equivalents were separated on 12% SDS-PAGE using standard procedures. PageRuler TM Prestained Protein Ladder (Fermentas, USA) was used as a protein standard. The proteins were transferred onto Odyssey Nitrocellulose Membranes (LI-COR Inc. Lincoln, Nebraska, USA). The membranes were dried in the air for 1 h and blocked at room temperature with 4% nonfat dry milk in 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 10% Tween-20 (TBST), and then incubated with mouse monoclonal antibody against the peptide sequence SEKDEL conjugated to KLH (Abcam Inc. Cambridge, MA, USA). Loading controls were performed using rabbit polyclonal antibody against full length native GAPDH protein from human erythrocytes (Abcam, USA) at 1:1000 dilution in 4% nonfat milk TBST solution at 4uC overnight. After washing with TBS containing 0.1% Tween-20 (TBST), the membranes were incubated with goat anti-rabbit IRDyeH 800CW or goat anti-mouse IRDyeH 800CW secondary antibody (LI-COR Biosciences, USA) at a dilution of 1:10000 in TBST/4% nonfat dry milk for 1 h at room temperature. Labeled proteins were visualized using Odyssey Infrared Imaging System (LI-COR Biosciences, USA).