Figure 3.

CHIP knockdown inhibits NF-κB and IRF3/7. (A) RAW264.7 cells stably transfected with scrambled control vector (RAW264.7-CTRL) or CHIP silencing vector (RAW264.7-CHIP RNAi) were treated with 100 ng/ml LPS or 1 µM CpG ODN for 1 h. Next, the intracellular translocation of p50, p65, IRF3, and IRF7 were evaluated by confocal microscopy using primary antibodies (Ab) specific for the indicated proteins and Oregon Green 488–conjugated secondary antibody. Bar, 150 µm. (B and C) Cells in A were lysed and nuclear extracts were isolated. Next, DNA-binding capacity of NF-κB was examined by EMSA (B), and the nuclear translocation of IRF3/7 was examined by Western blot (C). For supershift assays in B, 1 µg of control IgG, anti-p50, or anti-p65 antibodies, where indicated, were incubated for 30 min on ice before the adding of probes. (D and E) 48 h after transient transfection of siRNAs in day 6 BMDCs and day 10 pDCs, the cells were treated with indicated TLR agonists for 1 h, and nuclear extracts were used in EMSA assays (D) or examined for nuclear IRF3/7 by Western blot (E). The data shown correspond to a representative experiment out of three performed.