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).
Fig 2: Isolation of the BChE–protein complexes by size-exclusion chromatography (SEC) of serum proteins. (A) Chromatogram of human serum proteins separated on the TSK gel 3000 SWXL (Tosoh, Tokyo, Japan). Protein elution was monitored at 280 nm. The first 13 fractions of 200 µL were collected every 12 s in separate tubes. (B) Determination of the BChE activity in the fractions by Ellman’s assay in the collected fractions. The BChE activity is shown as dA412/min in red numbers above corresponding bars. dA412/min stands for change in absorbance at 412 nm per minute. (C) Analysis of the protein complexes in the 13 fractions by native polyacrylamide gel electrophoresis on Mini-PROTEAN 4–15% TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA). Ten microliters of each fraction was separated under native conditions in TB buffer at 4 °C. (D) SDS-PAGE analysis of the protein complexes in the 13 fractions. Ten microliters of each fraction sample was separated on Mini-PROTEAN 4–15% precast TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). M—protein marker; 1–13—10 µL of fractions. (E) Western blot analysis of 13 fractions using mouse monoclonal anti-BChE antibody (D-5): sc-377403 Santa Cruz Biotechnology (Santa Cruz, CA, USA) (1/200 dilution). (F) Western blot analysis of the fractions using rabbit monoclonal anti-ApoA-I antibody (ab52945) (Abcam, Cambridge, UK) (1/2000 dilution). (G) Western blot analysis of the fractions using rabbit monoclonal anti-ApoB antibody (ab139401) (Abcam, Cambridge, UK) (1/2000 dilution).
Fig 3: Validation of EV, high-density lipoprotein (HDL), and low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL) extraction methods. (A) Identities of EVs isolated from pooled human plasma and serum were determined by TEM. Representative images are shown. Orange arrows indicate the isolated EVs. (B) Expression levels of the EV markers (CD9 and MHC class I) and endoplasmic reticulum (ER) marker (calnexin) in the isolated EV samples examined by Western blot. HeLa cell lysate was using as a positive control. (C) Particle size distribution of the isolated EVs assessed by the NTA. Measurements were performed 10 times. The mean and mode ± SE of the calculated particle sizes are shown in the graph. (D) Expression levels of apoB-100 (LDL/VLDL marker) and apoA-I (HDL marker) in the isolated LDL/VLDL, HDL, and EV fractions, examined by Western blot.
Fig 4: Characterization of the HDL removal from the fractionated EV and yield/quality of the isolated EV.a Schematic of the EV fractionation and immunocapture of HDL. The diluted plasma (sample I) runs through the asymmetric nanoporous membrane to remove other particles with a size-exclusion mechanism, thus obtaining the EV fraction (sample II). Due to the dominating amount of HDL, a small portion of HDL remained in the EV fraction, so it was incubated with anti-ApoA1 and anti-ApoB antibodies, and then magnetic nanobeads conjugated with anti-rabbit IgG antibodies and run through the MNM device to remove the HDL, and the flow-through was collected (sample III). b Lipoproteins (cholesterol) remnant at different stages of purification from samples I, II, III in a), and (inset) EV concentration before and after MNM immunocapture (sample II and III) (n = 5 measurements for each sample). c Size distribution of EV samples with NTA before (sample II) and after MNM immunocapture (sample III) showing minimal EV loss or lysis/coalescence. (inset) CD63 and CD9 concentration before and after MNM immunocapture (sample II and III) measured by ELISA (n = 2 and 3 measurements for CD63, CD9, respectively). Error bars indicate the standard deviation (SD) in each plot.
Fig 5: Characterisation of PL-EVs. (A–G) PL-EVs isolated from control subjects (n = 3): (A) Representative TEM images from UC and SEC + UF extraction. Thirty thousand (30K) and 100,000 (100 K) magnifications are shown, white arrows indicate some of the PL-EVs presented in the samples. (B) Quantification of the number of particles/mL. (C) Size (diameter, mean) of particles and (D) size distribution of particles determined by NTA. (E) Quantification of total protein cargo and (F) ratio of protein per particle. (G) Western blot results from a control subject for platelet marker (CD61), EV markers (ALIX, CD63, CD9), and non-EV components (ApoB, Albumin). The numbers corresponding to the SEC + UF represent the first four fractions collected. (H–L) PL-EVs isolated by UC from controls and mild and severe patients: (H) number of particles/mL and (I) size (diameter, mean) of particles measured by NTA (n = 5–6); (J) particle protein concentration (n = 4–5); (K) ratio of protein:particle (n = 4–5); and (L) size distribution of particles determined by NTA (n = 5–6). Plts, platelets; UC, ultracentrifugation method; SEC + UF, size exclusion chromatography + ultrafiltration method; ApoB, Apoprotein B; ALIX, ALG-2 interacting protein X.
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