Fig 1: Fractionation of BChE–protein complexes by density iodixanol gradient ultracentrifugation (OptiPrep). (A) The separation procedure is summarized in the flow chart. (B) Picture of the centrifuged tube taken after separation. Positions of fractions are marked on the tube. (C) BChE activity of 10 uL of each fraction was estimated by Ellman’s assay as it has been described in Figure 1B. The BChE activity is shown as dA412/min. (D) Sudan black stained agarose gel electrophoresis profile of separated fractions after electrophoresis. Ellman’s reaction was developed in 100 mM PB buffer (pH 7.4) with a final concentration of 0.5 mM DTNB and 5 mM BTC. S—1 µL of serum; 4–15—1 µL of fractions. (E) SDS-PAGE; 1 µL of the fraction samples were separated on Mini-PROTEAN 4–15% precast TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). SDS-PAGE and Western blotting were performed according to the method described in Figure 1D. S—1 µL of serum; M—protein marker; 4–15—1 µL of fractions. (F) Western blot analysis of the fractions using rabbit monoclonal anti-ApoB antibody (ab139401) (Abcam, Cambridge, UK) (1/2000 dilution). (G) Western blot analysis of the fractions using rabbit monoclonal anti-ApoA-I antibody (ab52945) (Abcam, Cambridge, UK) (1/2000 dilution). (H) Western blot analysis of the fractions using mouse monoclonal anti-BChE antibody (D-5): sc-377403 Santa Cruz Biotechnology (Santa Cruz, CA, USA)(1/200 dilution). (I) Western blot analysis of the fractions using rabbit monoclonal anti-vitronectin antibody (ab46808) (Abcam, Cambridge, UK) (1/2000 dilution).