Fig. 1

PRAK interacts with the RAGE. a Yeast two-hybrid screening to identify RAGE interacting proteins in a human brain cDNA library. Yeast transformants of the RAGE bait and human brain cDNA library were spread on selection medium SD-LWU (SD without leucine, tryptophan and uracil), SD-LWA (SD without leucine, tryptophan and adenosine) and filter assay. pGBKT- PTB and pACT2-PTB served as the positive control (+). pGBKT and pACT2 were used as negative control (−). b PRAK and RAGE binding in vitro. Immunoprecipitation (IP) with anti-GFP antibody was accomplished using lysates from CHO cells transfected with RAGE and GFP-tagged PRAK, followed by western blotting with anti-RAGE antibody. c Binding kinetics of the RAGE C-term to PRAK protein. GST-fused PRAK protein was immobilized onto a CM5 sensor chip as the ligand. The RAGE C-term was used as the analyte from 0 to 40 μM to measure the kinetics of binding. Curves corresponding to multiple analyte concentrations were generated to ensure the precision of the calculation of the kinetics. The binding kinetics were analyzed using BIAevaluation 3.1 software