Silencing of CHIP impairs TLR-triggered innate response in macrophages and DCs. 48 h after transient transfection of 30-nM siRNAs in indicated cells, the cells were treated with 160 ng/ml Pam3Cys (Pam3), 10 µg/ml poly (I:C) (p[I:C]), 100 ng/ml LPS, 2 µg/ml R848, or 1 µM CpG ODN for 4 h (for macrophages) or 8 h (for DCs). Results were presented as mean concentration (picogram/milliliter) of indicated cytokines in the culture supernatants ± SD of triplicate samples. CTRL, scrambled control siRNAs; PM, peritoneal macrophages; BMDCs, BM-derived DCs; pDCs, plasmacytoid DCs; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. The data shown correspond to a representative experiment out of three performed.

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.

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.

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.

CHIP polyubiquitinates Src and PKCζ. (A and B) In vivo polyubiquitination assays. RAW264.7 cells stably transfected with scrambled control RNAi (CTRL) or CHIP-specific RNAi (RNAi) were treated with 100 ng/ml LPS or 1 µM CpG ODN for 30 min. Src and PKCζ contained in lysates were then subjected to heat denaturing in 1% SDS and immunoprecipitated (IP) with Src (A) or PKCζ (B) antibody plus protein A/G agarose. Ubiquitination was examined by immunoblot (IB) using anti-ubiquitin (Ub), anti–K63-Ub, or anti–K48-Ub antibody as indicated. (C) In vitro polyubiquitination assays. GST-CHIP was incubated with recombinant Src or PKCζ in the presence of E1, His6-Ub derivatives, and UbcH5A or Ubc13/Uev1A E2/Ubc as indicated. Src and PKCζ were immunoprecipitated (IP) with anti-Src or anti-PKCζ antibody after heat denaturing in 1% SDS. UbK63O, ubiquitin containing only Lys63; UbK48O, ubiquitin containing only Lys48. (D) Predicted ubiquitination sites in Src and PKCζ using the Bayesian discriminant method-prediction of ubiquitination sites algorithm, as well as protein sequence alignment. (E) RAW264.7 cells were transiently transfected with Flag-tagged wild-type or mutant Src (left) or PKCζ (right) constructs for 48 h, and then treated with 100 ng/ml LPS for 30 min. Ubiquitin levels associated with Src/PKCζ (mutants) were evaluated by ELISA at 450 nm. 1 ng recombinant ubiquitin-His was used as positive control. (F) RAW264.7 cells stably transfected with CHIP-HA were transiently transfected with Flag-tagged wild-type or mutant (KR1-3) Src or PKCζ constructs for 48 h, and then treated with 100 ng/ml LPS for 30 min. Src and PKCζ contained in lysates were immunoprecipitated (IP) with Flag agarose or IgG plus protein A/G agarose as indicated. Ubiquitination was examined by immunoblot (IB) using anti-Ub antibody. (G and H) RAW264.7 cells stably transfected with mock vector (RAW264.7-Mock) or CHIP-HA vector (RAW264.7-CHIP) were transiently transfected with Flag-tagged Src and PKCζ mutants (KR1-3) and NF-κB (G) or IFN-β (H) reporters for 48 h, and then treated with 100 ng/ml LPS or 1 µM CpG ODN for 4 h. Data are shown as mean ± SD of triplicate samples. **, P < 0.01; ***, P < 0.001 (as compared with corresponding RAW264.7-CHIP cells transiently transfected with mock vector and treated with LPS/CpG). In F–H, KR1 indicates for Src-K5/7/9R, KR2 for Src-K324/329R, and KR3 for PKCζ-K220/225R. The data shown in A–C and E–H correspond to a representative experiment out of three performed.

Silencing of CHIP impairs TLR signaling pathways. (A) RAW264.7 cells were transfected with plasmids encoding siRNAs, and selected with 600 µg/ml neomycin. The efficiency of silencing was evaluated by Western blot. CTRL, scrambled control RNAi; RNAi, CHIP-specific RNAi. (B and C) Reporter assays in RAW264.7 cells. Cells in A were transiently transfected with NF-κB or IRF3 reporters (B) or IL-6, CCL5, or IFN-β promoter reporters for 48 h, and then treated with 100 ng/ml LPS, 10 µg/ml poly (I:C), or 1 µM CpG ODN for 4 h. Results were expressed as fold induction relative to the luciferase activity in unstimulated cells transfected with control vector. Data are shown as mean ± SD of triplicate samples. ns, not significant; *, P < 0.05; **, P < 0.01. The data shown correspond to a representative experiment out of three performed.

CHIP binds HSC70, Src and PKCζ. (A) Wild-type RAW264.7 cells were treated with 100 ng/ml LPS, 10 µg/ml poly (I:C), or 1 µM CpG ODN, as indicated, and whole-cell lysates (WCL) were prepared. TLR4, TLR9, and TLR3 were immunoprecipitated (IP) with corresponding antibody plus protein A/G beads, and then components in immune complex were examined by Western blot. (B) GST fusion proteins were confirmed by immunoblot (IB) using GST antibody (top). GST pull-down assays were performed using lysates (WCL) from RAW264.7 cells (middle) or recombinant proteins (bottom). *, 30 µg lysates; **, 1 ng recombinant proteins. (C) HEK293 cells were co-transfected with plasmids encoding HA-tagged CHIP or mutants and plasmids encoding Flag-tagged target molecules. 48 h later, immunoprecipitation assays were performed using anti-HA agarose or IgG agarose (*). The data shown correspond to a representative experiment out of three performed.

CHIP promotes TLR-triggered Src- and PKCζ-dependent activation of IκBα, IRAK1, and TBK1. (A) Western blot assays of signaling mediators in RAW264.7 cells stably transfected with mock control vector or HA-tagged CHIP vector (CHIP-HA) and treated with 100 ng/ml LPS or 1 µM CpG ODN, as indicated. Otherwise, cells were pretreated with 10 nM Src inhibitor (PP1) or PKCζ 10 µM pseudosubstrate peptide (PS) for 30 min before LPS or CpG stimuli. (B and C) In vitro kinase assays of IRAK1 and TBK1. In B, RAW264.7 cells stably transfected with mock control vector or HA-tagged CHIP vector (CHIP-HA) were used. In C, RAW264.7 cells stably transfected with scrambled control RNAi (CTRL) or CHIP-specific RNAi (RNAi) were used. These cells were treated with 100 ng/ml LPS or 1 µM CpG ODN for 30 min. Kinase activity of immunoprecipitated (IP) IRAK1 and TBK1 was evaluated by in vitro kinase assays in the presence of 1 µCi of [γ-³²P]-ATP (6,000 Ci/mmol) and 0.5 µg of MBP as substrate. (D and E) In vitro kinase assays of IRAK1 and TBK1. Cells in A were transiently transfected with mock vector or Flag-tagged SrcY416F or PKCζT410A mutant vector for 48 h. After treatment with 100 ng/ml LPS or 1 µM CpG ODN for 30 min, the kinase activity of immunoprecipitated (IP) IRAK1 and TBK1 was evaluated by in vitro kinase assays in the presence of 1 µCi of [γ-³²P]-ATP (6,000 Ci/mmol) and 0.5 µg of IRF3 (D) or IRF7 (E) as substrates. In B–E, results were presented as mean radioactivity ± SD of triplicate samples. ns, not significant; *, P < 0.05; **, P < 0.05; ***, P < 0.001. The data shown correspond to a representative experiment out of three performed.

CHIP colocalizes with Src and PKCζ. (A) Localization of CHIP in endosomes. RAW264.7 cells were transiently transfected with GFP-tagged CHIPΔN20 or CHIP vector for 48 h. Cells were stained with anti-EEA1 and Alexa Fluor 555–conjugated secondary antibody. Bars, 100 µm. (B) RAW264.7 cells were transiently transfected with CHIP-GFP for 48 h, and treated with 100 ng/ml LPS for 10 min (rows 1–3). Cells were stained with anti-TLR4, Src, PKCζ antibodies, and Alexa Fluor 555–conjugated secondary antibody. RAW264.7 cells were treated with 100 ng/ml LPS for 10 min (rows 4 and 5). Cells were stained with anti-TLR4, Src, or PKCζ antibodies, and Oregon Green 488-conjugated or Alexa Fluor 555–conjugated secondary antibody. In B, the merged images are shown. The boxed regions in the middle image were correspondingly magnified and shown on the right. White arrow heads indicated for the typical colocalization sites. Bars, 100 µm. The data shown correspond to one representative experiment out of three performed.

CHIP enhances TRL4- and TLR9-triggered DC maturation via IFN-α/β–dependent pathway. (A) 48 h after transient transfection of siRNAs in day 6 BMDCs, the cells were treated with 100 ng/ml LPS or 1 µM CpG ODN for 4 h. The mRNA levels of IL-12p40 and CD40 were evaluated by quantitative PCR. (B) Day 6 BMDCs were transiently co-transfected with mock vector, CHIP-HA or CHIP mutant-HA vectors, and SrcY416F or PKCζT410A vector as indicated for 48 h, and then treated with 100 ng/ml LPS or 1 µM CpG ODN for 8 h in the presence or absence of 10 nM Src inhibitor (PP1) or 10 µM PKCζ pseudosubstrate peptide (PS). IFN-β levels were measured by ELISA. In A and B, results were presented as mean ± SD of triplicate samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C and D) Day 6 BMDCs derived from wild-type or IFNAR^−/− mice were transiently transfected with mock vector or CHIP-HA vector for 48 h, and treated with 100 ng/ml LPS or 1 µM CpG ODN for 48 h. The expression levels of DC markers CD40 (C) and CD80 (D) were evaluated by FACS assays, and the numbers in each panel indicate mean fluorescence intensity. The data shown correspond to one representative experiment out of three performed.