Journal: Frontiers in Endocrinology
Article Title: Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains
doi: 10.3389/fendo.2024.1451279
Figure Lengend Snippet: DOC2B is detectable in human plasma EVs. (A) Schematic representation of DOC2B domains (top). Highlighted region in red represents the region targeted by the in-house DOC2B antibody #2; the epitope of the DOC2B protein (amino acids 96-116). 3D model structure of human DOC2B protein (bottom), generated by I-TASSER. The epitope of the DOC2B antibody is superimposed in Pymol. The epitope is shown in red color. (B) Immunoblot (IB) validation of the specificity of the in-house DOC2B antibody #2 in recognizing recombinant human DOC2B protein (hDOC2B) via epitope-specific blocking peptides (BP); Black vertical dashed lines indicate splicing of lanes from within the same gel exposure. Representative of n=2 independent measurements. (C) Schematic of the EV isolation from pooled human plasma from individuals with no diabetes (ND) using 70 nm size exclusion chromatography (SEC) column. Created with Biorender.com . Representative dotblots below the schematic show protein detection of the EV markers TSPAN and SYN; DOC2B; protein that often co-isolates with EVs ApoA; and Cyt. C in the SEC fractions F1-F6. Void volume (VV) is negative control. Representative of n=3 independent measurements. (D) Size distribution of the combined SEC fractions F1-F5 acquired using NTA (mean ± SEM, n=3 independent measurements). The average total EV concentration was calculated to be 3.5 x10 9 particles/mL (considering all detected EV sizes) and the average diameter was ~160 nm. (E) TEM image of the combined SEC fractions F1-F5 indicates intact EVs, pointed with yellow arrows. The image was taken at 30,000x magnification. Representative of n=3 independent measurements. (F) Protein levels for the EV marker CD81 and DOC2B were obtained for plasma EVs (SEC F1-F5 combined) and extracellular soluble protein (SEC F12-F13 combined) via immunoblot using antibodies against CD81; DOC2B (in-house DOC2B antibody #2); Ponceau S served as loading control; 3 independent ND human plasma EV samples were assessed. Black vertical dashed lines indicate splicing of lanes from within the same gel exposure.
Article Snippet: To isolate EVs, 0.4 mL of plasma was passed through a qEV original/70 nm Legacy [Izon Science (Portland, OR, USA), Cat# SP1] size exclusion chromatography (SEC) column as indicated by manufacturer instructions.
Techniques: Clinical Proteomics, Generated, Western Blot, Biomarker Discovery, Recombinant, Blocking Assay, Isolation, Size-exclusion Chromatography, Negative Control, Concentration Assay, Marker, Control
