Fig. 1.

P2X7R inhibition blocks TG2 secretion by macrophages. (A) Differentiated monocytes express TG2. THP-1 cells were differentiated for the indicated time with TPA and stimulated with LPS as indicated. Cell extracts were analyzed by western blotting for TG2 or β-tubulin as a loading control (*, non-specific reactivity). (B) TG2 export requires P2X7R activity. Differentiated THP-1 cells were pre-treated with vehicle or 5 μM P2X7R inhibitor A740003 for 10 min, then stimulated as indicated with BzATP for 10 min with or without inhibitor (inh, pulse). Cells were chased for 30 min in P2X7R agonist and antagonist-free medium. Collected medium from the pulse and chase (200 µl) were rendered cell-free by centrifugation, and analyzed for TG2 by western blotting. (C,D) P2X7R activation triggers TG2 secretion in macrophages derived from peripheral blood mononuclear cells. Macrophages were stimulated with BzATP and chased as in B, and collected medium from the of pulse and chase were analyzed for TG2 by western blotting alongside the cell lysates (C). The presence of 100 µM Ac-YVAD-CMK did not prevent externalization or cleavage of TG2, indicating a caspase-1-independent process (D).