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. Author manuscript; available in PMC: 2021 Jun 11.
Published in final edited form as: Biochemistry. 2020 May 20;59(21):1981–2002. doi: 10.1021/acs.biochem.0c00274

Figure 2.

Figure 2.

Increasing concentrations of IGFBP-3 bind HN and abolish its binding to Aβ in the absence or presence of AChE. (A) Interaction of Aβ40 (■) or Aβ42 (●) with biotinylated HN. Aβ (100 nM) was bound to the wells. Increasing concentrations of biotinylated HN were then added and processed as described in the experimental procedures. Optical density measurements (450 nm) were normalized by expressing each point in relation to the best-fit Emax value (set to 100%). The data were then plotted as a function of the increasing biotinylated HN concentrations. Using GraphPad Prism 8.3.1, the data were fit to a single binding site model with a nonlinear regression curve fitting approach and plotted as the mean ± SD of three independent trials, each of which was run in triplicate. (B and C) Competition of IGFBP-3 for the complex between HN and Aβ40 or Aβ42. Aβ (100 nM) was bound to the plate wells. Then, a single concentration of biotinylated HN (100 nM, ●) or a combination of HN and AChE [biotinylated HN (100 nM) + AChE (100 nM)] (■) was incubated for 1 h with or without increasing concentrations of IGFBP-3 and loaded into the Aβ coated wells. The signal was then processed as described in the Experimental Procedures. Arrows on the x-axis indicate the IGFBP-3 concentration corresponding to 50% inhibition for each of the following curves: Aβ40 (●, 93 ± 16 nM), Aβ40 (■, 74 ± 14 nM), Aβ42 (●, 58 ± 11 nM), and Aβ42 (■, 39 ± 7 nM). The dashed line indicates 50% of the maximum binding. Prior to data analysis, the OD was corrected for nonspecific binding by subtracting the mean background absorbance for the negative controls prepared with all components except biotinylated HN. Data were then normalized by plotting the mean absorbances for each concentration as a fraction of the maximal binding (set to 100%) and as a function of the IGFBP-3 concentrations. Using GraphPad Prism 8.3.1, the data were analyzed with a nonlinear regression curve fitting approach then expressed as the mean ± SD of three independent experiments, each of which was carried out in triplicate.