Fig. 2

RBGO1 antibody, targeting the V-region of RAGE is internalized more rapidly than antibodies targeting other regions of the RAGE protein and binds with higher affinity to whole RAGE protein. Schematic diagram of the relative binding positions on the RAGE protein of each of the 4 antibodies tested (a). HEC1A endometrial cancer cells were treated with control medium or medium containing monoclonal antibodies against RAGE at 37 °C for 1 h. After incubation, the cells were washed, fixed and permeabilized. Internalized antibody: RBGO1 (b), RBGO2 (c), RBGO3 (d) or RBGO4 (e), was imaged via fluorescently labelled secondary antibodies and nuclei stained with DAPI. Cells were also incubated only with the secondary antibody as negative control (f). Images were acquired on a Zeiss LSM 710 confocal microscope and analyzed using the Zen 2012 image analysis software. The quantity of internalized antibody was determined using Image J software as a function of cell area (g). For antibody binding kinetics (h), antibodies were captured to a Sensor Chip CM5 via an amine coupled anti-mouse antibody followed by single-cycle kinetics experiments. RBGO1, RBGO2, RBGO3 or RBGO4 antibodies were exposed to whole RAGE protein (2.5 to 200 nM) and data were fitted using a one-to-one Langmuir binding model. Data are expressed as mean (SD) from 3 independent experiments. Data were analyzed by ANOVA and Dunnett’s multiple comparison test. RBGO1 differs from each of the other antibodies, ***p < 0.001.