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. 2011 Sep 26;208(10):2099–2112. doi: 10.1084/jem.20102667

Figure 7.

Figure 7.

Knockdown of Src and PKCζ blocks the effects of CHIP in TLR response. (A) RAW264.7 cells stably transfected with mock vector or CHIP-HA vector were transiently transfected with control siRNAs (CTRL, for GFP) or Src/PKCζ-specific siRNAs for 48 h. The efficiency of silencing was evaluated by Western blot. (B) Cells in A were transiently transfected with NF-κB reporters for 48 h. Cells were treated with 100 ng/ml LPS or 1 µM CpG ODN for 4 h. Results were expressed as fold induction relative to the activity in unstimulated cells transfected with mock vector. Data are shown as mean ± SD of triplicate samples. si-Src, siRNAs specific for Src; si-PKCζ, siRNAs specific for PKCζ; ns, not significant; **, P < 0.01; ***, P < 0.001 (as compared with corresponding cells without LPS/CpG treatments). (C and D) Cells in A were treated with 100 ng/ml LPS for 1 h (C) or 30 min (D), and then nuclear extracts (C) and cell lysates (D) were prepared. Nuclear translocation of IRF3/7 (C) or phosphorylated IRAK1/TBK1 within immunoprecipitates (IP; D) was examined by Western blot. s-1, control siRNAs for GFP; s-2, Src-specific siRNAs; s-3, PKCζ-specific siRNAs. The data shown correspond to a representative experiment out of three performed.