Fig. 6: BIG1 deficiency reduced PIP2 production and prohibited the binding of TIRAP to the plasma membrane of macrophages induced by LPS.

a WT and BIG1^−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min. The cell lysates were subjected to immunoblotting with the indicated antibodies. b The membrane-bounded and cytosolic proteins from WT and BIG1^−/− BMDMs stimulated with LPS (100 ng/ml) for 30 min were measured by immunoblotting with the indicated antibodies. c WT and BIG1^−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min, followed by confocal imaging analysis with TIRAP antibody. Scale bars, 20 μm. d WT and BIG1^−/− BMDMs were treated with LPS (100 ng/ml) for 30 min, followed by dot blot with the indicated antibodies (up panel). The activity of PIP5K was measured by luminescence assay kit (down panel). e The cell surface proteins from WT and BIG1^−/− BMDMs treated with LPS (100 ng/ml) for 30 min, were measured by dot blot and immunoblotting with the indicated antibodies, respectively. f WT and BIG1^−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min, followed by confocal imaging analysis with PIP2 antibody. Scale bars, 20 μm. g PIP2 fluorescent intensities from more than 50 individual cells were quantified in f. h WT and BIG1^−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min, then cellular PIP2 levels from extracted acidic lipids were measured by using the PIP2 mass ELISA kit. i, j BIG1^−/− BMDMs transfected with active mutant ARF3 (Q71L) or vector were treated with or without LPS (100 ng/ml) for 30 min, followed by dot blot and Western blot with the indicated antibodies (i) or measuring the activity of PIP5K (j). k THP-1-derived macrophages transfected with negative control siRNA or si-BIG1 were treated with or without LPS (100 ng/ml) for 30 min. The membrane-bounded and cytosolic proteins were isolated, followed by dot blotting with the indicated antibodies. Data show pooled technical replicates from three independent experiments (d, g, h, j). All immunoblot data are representative of three independent experiments with similar results. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (one way ANOVA in d, g, h, j).