Assessment of FL-RAGE expression and cRAGE production

Two thousand R3/1-pLXSN or R3/1-FL-RAGE clones were seeded on 6 well-plate (Costar, Kennebunk, ME, USA) and treated with indicated concentration of human glycated Albumin (HGA; #A8301, Sigma Aldrich, Saint Louis, Missouri, USA) or not-glycated human Albumin (HA; #A7736, Sigma Aldrich) as control in DMEM supplemented with 0.1% FBS for 6 hours. Supernatant of cells was tested for cRAGE concentration by a commercial ELISA (DY1145, Human RAGE DuoSet ELISA, R&D Systems Inc., MN, USA). Cells were harvested in RIPA buffer in the presence of proteases inhibitors (P8849, Sigma-Aldrich), incubated in ice for 30 min and centrifuged at 12000g for 15 minutes at 4°C. Protein extracts were separated by SDS-PAGE and transferred to Amersham Hybond ECL Nitrocellulose membranes (GE Healthcare, Little Chalfont, United Kingdom). Membranes were incubated with a goat anti-human RAGE (1 µg/ml; cat. AF1145; R&D Systems Minneapolis, MN, USA) or an antibody against GAPDH (0.2 µg/mL, sc-25778, Santa Cruz Biotechnology) as a loading control. Proteins were visualized by an enhanced chemiluminescence (ECL) detection system (cat. RPN2106, GE Healthcare) and acquired with a ChemiDoc™ MP Imaging System (Biorad, Hercules, CA, USA). Protein bands were quantified by densitometry analysis using ImageJ.