Fig. 4: Corisin interacts with serum human albumin.

A, B The predicted interaction structure of human serum albumin-corisin and bovine serum albumin-corisin by molecular dynamic simulations. Human serum albumin and bovine serum albumin are shown in surface representation, and domains I, II, and III are shown in pink, yellow, and ice blue, respectively. The corisin is shown in licorice representation. C, D Western blotting showing the interaction of corisin with recombinant human albumin (rhAlb) in vitro. A fixed concentration of rhAlb was combined with concentrations of synthetic corisin or scrambled peptide and incubated at room temperature for 10 min. The mixture underwent sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting using anti-corisin monoclonal antibody (mAb) or anti-rhAlb antibody. The experiment was independently repeated three times with similar results. E Co-immunoprecipitation of corisin with rhAlb. Synthetic corisin was incubated with rhAlb, treated with an anti-corisin mAb, and immunoprecipitated using protein G agarose beads. rhAlb was not immunoprecipitated by the anti-corisin mAb and protein G agarose beads alone (lane 5). In the presence of corisin, rhAlb was successfully co-immunoprecipitated by the anti-corisin mAb and protein G agarose beads (lane 6). The experiment was independently repeated three times with similar results. Ig, immunoglobulin; MW, molecular weight. F Corisin interacts with human and mouse serum albumin. A predetermined concentration of human serum albumin (HSA) or mouse serum albumin (MSA) was incubated with synthetic corisin. The mixture was subjected to SDS-PAGE and Western blotting using an anti-corisin monoclonal antibody. The experiment was independently repeated three times with similar results. G Corisin interacts with human serum albumin derived from both patients with diabetic nephropathy and healthy individuals. Serum samples were diluted (1:20) and incubated for 10 min with corisin (4 µg) diluted in saline. As a control, a mixture containing corisin (4 µg) and rhAlb (5 µg) was prepared. Each mixture was then subjected to electrophoresis, followed by Western blot analysis using mAb specific for corisin or human albumin. The experiment was independently repeated three times with similar results. H, I Complex formation between corisin and albumin in the urine of diabetic CKD patients. Urine samples from 3 diabetic patients with chronic kidney disease (CKD) and 3 healthy subjects were concentrated 5 times, and 5 µL of each sample were subjected to SDS-PAGE, followed by Western blotting using an anti-corisin mAb (left panel) or anti-human albumin antibody (right panel). DM diabetes mellitus.