(A–C) BMMs were primed overnight with LPS and then transfected with ultrapure S. minnesota RE595 LPS packaged with DOTAP.
(A) Immunoblotting for pannexin-1 before and after LPS transfection in wild-type (C57BL/6), Casp11−/− and Panx1−/− BMMs.
(B) Pannexin-1 cleavage after LPS transfection in Casp11−/− BMMs reconstituted with lentiviral vectors expressing wild-type caspase-11, caspase-11 C254S and vector alone. Immunoblotting for caspase-11 and β-actin are also shown.
(C) ATP release (upper panel), cytotoxicity (middle panel) and pannexin-1 cleavage (lower panel) were measured after LPS transfection in Panx1−/− BMMs reconstituted with wild-type pannexin-1, pannexin-1 D378A lentiviruses or control vector. Immunoblotting for pannexin-1 and caspase-11 in wild-type BMMs is also shown as a control.
(A–C) Data are representative of at least 3 experiments. Graphs show mean ± SD of triplicate cultures, * p <0.05. See also Figure S6.