SHIP-1 decreases NOD2-induced NF-κB activation in epithelial cells.

<p><b>A.</b> HEK293T cells were transfected with plasmids encoding a NF-κB luciferase reporter gene and NOD2 along with increasing amounts of SHIP-1. Positive control was achieved by treating cells transfected with SHIP-1 or with an empty vector with TNF-α (250 U/mL) for 6 h. NF-κB activity was measured by luciferase assay 36 h post transfection (mean ± S.D (n = 3), *p<0.01). <b>B.</b> GNV cells were transfected with a NF-κB luciferase reporter gene along with increasing amounts of SHIP-1. Cells were subsequently treated with MDP (1 µg/mL) for 18 h or left untreated. NF-κB activity was determined by luciferase assay (mean ± S.D (n = 3), *p<0.04). <b>C.</b> GNV cells were transfected with 2.5 µg of either a SHIP-1 or an empty vector. 24 h post transfection, cells were treated with MDP (50 µg/mL) for indicated periods or with TNF-α (250 U/mL) for 1 h. RNA was extracted and <i>il-8</i> mRNA level was measured by quantitative real-time PCR (mean ± S.D (n = 3), *p<0.001). <b>D.</b> GNV cells were transfected with 2.5 µg of either a SHIP-1 or an empty vector. 24 h post transfection, cells were treated with MDP (50 µg/mL) for indicated periods and proteins were extracted. Samples were immunoblotted with an anti-SHIP-1 and an anti-p-IκBα antibody. Actin was used as loading control.