Fig. 4.

P2X7R-mediated TG2 export is not due to microvesicle release. (A,B) Analysis of vesicle release by nanoparticle tracking. TG2- or mock-transfected P2X7R cells were stimulated with BzATP for 10 min, chased for 30 min in agonist-free medium, and conditioned media were analyzed for nanoparticles using light scattering in combination with particle tracking (Nanosight). Particle distribution and total particle concentration is shown (mean±s.e.m.; n=5) (A). Particles were broadly assigned to one of four fractions based on volume: representing exosomes (∼60 nm; ≤80 nm diameter), microvesicles (∼145 nm; 81–262 nm), larger vesicles (∼335 nm; 263–425 nm) and aggregates or membrane blebs (≥426 nm) (B). (C,D) Analysis of isolated microvesicles for TG2. Cell-free medium (S1) from BzATP- or control-treated cells was subjected to differential centrifugation (P, pellet; S, supernatant): in C, 3000 g twice (P2, P3), 10,000 g (P4), and 100,000 g (P5, S5), and in D, 3000 g followed by separation of microvesicles (MV) on a sucrose cushion. Fractions were analyzed for TG2 by western blotting.