Fig. 5.

Extracellular Ca²⁺ regulates TG2 secretion. (A) P2X7R-mediated TG2 export at different [Ca²⁺]_ex. P2X7R cells expressing TG2 were stimulated with BzATP for 10 min in medium containing 0.9 or 2.2 mM Ca²⁺ or in Ca²⁺-free medium, and chased for 30 min in respective media without BzATP. Conditioned media were analyzed by western blotting for TG2 and flotillin-2. (B) TG2 catalytic activity is not required for P2X7R-mediated export. P2X7R cells expressing TG2 or the TG2 C277S mutant were stimulated with BzATP in medium containing 0.9 or 2.2 mM Ca²⁺ and TG2 export was assessed as above. (C,D) [Ca²⁺]_ex regulates P2X7R activity. P2X7R or parental cells were stimulated with BzATP, as indicated, in PSS containing YO-PRO1 and different concentrations of Ca²⁺. To determine YO-PRO1 uptake by cells after BzATP application, changes in well-specific fluorescence (λ_ex, 480-10 nm; λ_em, 520-10 nm) were monitored over time. A representative experiment of dye uptake in Ca²⁺-free PSS is shown as mean±s.e.m. of two wells (C). In D, the initial rates of YO-PRO1 uptake at different [Ca²⁺]_ex in response to 300 μM BzATP are given (mean±s.e.m.; n=2).