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 sorted into the lumen of β-cell EVs and its tandem C2s domain acts as an EV sorting signal. (A) Schematic of the permeabilization of INS-1 832/13-derived EVs with Tween 20 to expose luminal contents (top). Created with Biorender.com. The INS-1 832/13 SEC fractions F3-F5 were combined and dotblot at 10, 20, 100, and 200 ng of protein. The membranes were blocked with 50 mg/mL non-fat milk in tris buffered saline (TBS) without or with 0.2% Tween 20. Antibodies against DOC2B (in-house DOC2B antibody #2), TSG101 (a luminal EV protein), and the extracellular domains of TSPANs (membrane surface-associated EV protein) were prepared in the blocking solution and applied to the membranes overnight. Representative of n=3 independent measurements. (B) Protein levels of fusion proteins rDOC2B-GFP, rC2AB-GFP, and GFP vehicle in INS-1 832/13 cells transfected with plasmid DNA using Lipofectamine 2000 reagent, present in 35 nm SEC isolated EVs and extracellular soluble protein from the INS-1 832/13 CM and WCL, were assessed via immunoblot and probed with GFP antibody. * indicates rDOC2B-GFP. Ponceau S served as loading control for extracellular soluble protein and EVs, and GAPDH as loading control for WCL. Representative of n=4 independent measurements.

Article Snippet: Briefly, all antibodies were prepared in TBS supplemented with 0.2% Tween 20 (TBST) [G Biosciences (St. Louis, MO, USA), Cat# DG011].

Techniques: Derivative Assay, Saline, Membrane, Blocking Assay, Transfection, Plasmid Preparation, Isolation, Western Blot, Control