Nucb2 (Nucleobindin-2), a calcium-binding protein, is further cleaved into NES1 (Nesfatin-1). NES1 is an anorexigenic peptide and seems to participate in hypothalamic pathways regulating food intake and energy homeostasis, acting in a leptin-independent manner. GADD45A (Growth arrest and DNA damage-inducible protein GADD45 alpha) has been identified as an interactor of Nucb2. Besides, Growth Arrest And DNA Damage Inducible Protein Beta (GADD45b) has been identified as an interactor of NES1, thus a binding ELISA assay was conducted to detect the interaction of recombinant rat NES1 and recombinant rat GADD45b. Briefly, NES1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to GADD45b-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NES1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of NES1 and GADD45b was shown in Figure 1, and this effect was in a dose dependent manner.
Figure. The binding activity of NES1 with GADD45b.