Figure 8.

Involvement of RalB in inflammasome triggered autophagy. (a) Immunoblot of GTP-bound RalB collected by RALBP1-agarose affinity purification from cell lysates from LPS primed (20 ng/ml) mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM),1.5 μg/ml poly(dA-dT), or uric acid crystals (50 μg/ml). Immunoblotting visualized RalB-GTP in the pull downs and RalB in cell lysates. (b) Immunoblot of lysates and supernatants prepared from mouse BMDM transfected with RalB or control siRNA on 2 sequential days, primed with LPS and exposed to 1.5 μg/ml poly(dA-dT) for 6 h for RalB, LC3, actin, and IL-1β expression. To collect cell supernatants the cells were cultured overnight in serum free media. (c) Immunoblot of cell lysates from mouse BMDMs transfected with AIM2 or control siRNA on 2 sequential days, primed with LPS and 2h later exposed for 1h to poly(dA-dT) for the indicated proteins. A similar experiment using Aim2^−/− BMDMs is shown. (d) ASC immunoprecipitates from LPS primed mouse BMDM transfected with poly(dA-dT) for 1h, treated with LPS+ATP for 30 minutes, or non-stimulated immunoblotted as indicated. (e) Confocal microscopy of LPS primed mouse BMDMs transfected with poly(dA-dT) for 30 minutes were immunostained for RalB and AIM2 (first three panels); RalB and ASC (4^th and 5^th panels); and AIM2 and Beclin-1 (6^th and 7^th panel). Areas subjected to 3X electronic zoom are outlined by white squares. Scale bars shown are 10 μm. All experiments performed at least twice.