Fig. 2.
P2X7R activation mediates TG2 externalization. (A) Analysis of TG2 secretion in HEK293 P2X7R cells. TG2-transfected cells were stimulated with BzATP or vehicle for indicated time (pulse), then incubated for 30 min in agonist-free medium (chase). TG2 secretion into cell-free supernatants was assessed by western blotting. (B) Inhibitor A740003 reversibly blocks P2X7R activation. P2X7R cells were incubated with Fluo-4-AM and 5 μM P2X7R inhibitor for 20 min prior to BzATP stimulation in the presence of inhibitor (top), washed with inhibitor-free medium for 5 min, and then re-stimulated with BzATP (bottom). The fluorescence (λex, 488 nm; λem, 500–535 nm) change in individual cells was monitored by confocal microscopy (mean±s.e.m., n=30) (right). Optical sections of the same field before and 180 s after BzATP addition are shown (left). Scale bar: 25 µm. (C) P2X7R inhibitor (inh) blocks TG2 secretion. TG2-transfected P2X7R cells were pre-treated with P2X7R inhibitor or vehicle for 10 min before BzATP stimulation as indicated. TG2 release into medium was assessed as in A. (D,E) Cells release membrane-bound particles upon P2X7R activation. TG2 transfected P2X7R or parental cells were BzATP stimulated for 10 min, and chased in agonist-free medium. Conditioned media and cell lysate were analyzed by western blotting for TG2 and the microvesicle marker flotillin-2 (D) or, as a control, β-tubulin, IκBα and HMGB-1 (E).