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

Figure 5.

Figure 5.

CHIP knockdown inhibits TLR4/9-induced recruitment and activation of Src and PKCζ. (A) RAW264.7 cells stably transfected with CHIP-specific RNAi (RNAi) or scrambled control RNAi (CTRL) were treated with 100 ng/ml LPS or 1 µM CpG ODN, as indicated. TLR4 and TLR9 were immunoprecipitated (IP) with corresponding antibody plus protein A/G beads. Next, components in immune complex were examined by Western blot. WCL, whole-cell lysates. (B) Cell lysates derived from cells in A were immunoprecipitated with Src or PKCζ antibody or IgG plus protein A/G beads. Activation of Src (p-Src, phospho-Src) and PKCζ (p-PKCζ, phospho-PKCζ) was evaluated by immunoblot (IB) using phospho-specific antibodies. (C) RAW264.7 cells stably transfected with mock control vector or HA-tagged CHIP vector (CHIP-HA), pretreated with or without 10 nM Src inhibitor (PP1) or 10 µM PKCζ pseudosubstrate peptide (PS) for 30 min, were treated with 100 ng/ml LPS or 1 µM CpG ODN, as indicated. Activation of Src and PKCζ were evaluated as described in B. The data shown correspond to a representative experiment out of three performed.