Fig. 2: BIG1 only responded to LPS stimulation in BMDMs and THP-1-derived macrophages.

a WT BMDMs were stimulated with LPS (100 ng/ml) for 12 h, R848 (10 nM) for 24 h, CpG ODN (5 µM) for 24 h, the indicated proteins were analyzed by Western blot. b WT and BIG1^−/− BMDMs were stimulated with LPS (100 ng/ml) for 12 h, R848 (10 nM) for 24 h, CpG ODN (5 µM) for 24 h. After treatment, the levels of TNF-α, IL-6, and IL-1β mRNA were measured by RT-qPCR. c THP-1 cells were treated with LPS (100 ng/ml) for 12 h, R848 (10 nM) for 24 h, CpG ODN (5 µM) for 24 h after incubating with PMA (100 ng/ml) for 24 h, the indicated protein were analyzed by Western blot. d, e THP-1-derived macrophages transfected with negative control siRNA (NC) or BIG1 siRNA (SiRNA) were untreated or treated with LPS (100 ng/ml) for 12 h, R848 (10 nM) for 24 h, CpG ODN (5 µM) for 24 h. Total RNA was extracted. The levels of BIG1, TNF-α, IL-6, and IL-1β mRNA were measured by RT-qPCR. Data show pooled technical replicates from three independent experiments (b, d, e). 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 (Student’s t-test in b, d, e).