IKK recruitment to the CD40 signaling complex is defective in HOIP-deficient cells.

<p>(A) SMAC peptide treatment reduces recruitment of cIAP1 to CD40 and may modify HOIP recruitment. A20.2J cells were incubated for six hours with membrane-permeable SMAC-N7 peptide or 1.5% DMSO (solvent used for the peptide). Following the incubation, cell lysates were prepared, fractionated by SDS-PAGE, and evaluated by Western blot (lanes 1 and 2). Cells incubated with DMSO or SMAC-N7 were also stimulated with magnetic beads coated with anti-CD40 or an isotype control antibody. Immunoprecipitated (IP) material bound to the beads was loaded in lanes 3-5. Samples of the cell lysates after immunoprecipitation appear in lanes 6–8. Western blots were probed with antibodies specific for cIAP1, TRAF2, TRAF3, and HOIP (approximate molecular weights indicated on left). Similar results were obtained in two additional experiments. (B) CD40 was isolated by immunoprecipitation (as in (A)) from A20.2J cells and HOIP-deficient cells transduced with an empty retroviral vector (pMIP) or a retroviral vector encoding HOIP. Material immunoprecipitated with an isotype control antibody (isotype) or anti-CD40 antibody was examined by Western blotting for CD40, TRAF2, TRAF3, cIAP1, HOIP, IKKα/β, and IKKγ (right panels). HOIP expression was required for coprecipitation of IKK proteins with CD40. Cell lysates from unstimulated cells are shown in the left panels. Similar results were obtained in a second experiment and in two experiments with a second HOIP-deficient clone. (C) To further evaluate HOIP-dependent recruitment of IKKγ to CD40, A20.2J cells or HOIP-deficient (HOIP^-/-) A20.2J cells were transduced with an empty retroviral vector or a retroviral construct encoding BP epitope-tagged IKKγ (noted in the figure as pMIP and IKKγ, respectively). Lysates (lanes 1–3) and immunoprecipitation (IP) samples (lanes 4–11) from the cell lines were fractionated by SDS-PAGE and evaluated by Western blotting with antibodies to the BP tag (IKKγ, upper panel) and TRAF2. The anti-CD40 IP sample in lane 11 was treated with λ phosphatase; the sample in lane 10 was mock-treated. Protein samples (minus those treated with phosphatase) were also fractionated on a separate gel (lower acrylamide concentration) for the evaluation of TRAF3 and HOIP (bottom two panels). Similar results were obtained in two additional experiments.</p

CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.

<p>Cells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP^-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment.</p

HOIP gene targeting.

<p>(A) Regions of <i>Rnf31</i> gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with <i>Rnf31</i> resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP^-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of <i>Rnf31</i> (HOIP^−/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD.</p

GLε transcription is defective in CD40-stimulated HOIP-deficient cells.

<p>A20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP^-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP^-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.</p

CD40-induced activation of NF-κB and JNK is defective in HOIP-deficient cells.

<p>CD40-mediated signaling was defective in HOIP-deficient cells (HOIP^-/- + pMIP), but intact in parental cells transduced with an empty retroviral vector (A20.2J + pMIP) and in HOIP-deficient cells transduced with a retroviral vector encoding HOIP (HOIP^-/- + HOIP). Cells were activated with CD154-expressing insect cells for the times indicated. As a negative control, cells were stimulated with insect cells lacking CD154 (5 minute time point only). Western blots of whole-cell lysates were probed with the indicated antibodies. Phospho-IκBα (pIκBα) and phospho-JNK (pJNK) blots were stripped and reprobed for total IκBα and total JNK, respectively (anti-JNK antibodies recognize p46 and p54 isoforms). IκBα blots were also reprobed for actin to demonstrate equal lane loading. Molecular weights are indicated at right. Similar results were obtained in a second experiment and in two additional experiments with a second HOIP-deficient clone.</p

TRAF-dependent recruitment of proteins to CD40.

<p>Endogenous CD40 from CH12.LX cells (WT), TRAF2-deficient CH12.LX (T2-), or TRAF3 deficient CH12.LX cells (T3-) was isolated using ARC (lane loading as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011380#pone-0011380-g002" target="_blank">Fig. 2</a>). Western blotting was performed with the indicated antibodies. Each blot is representative of three or more experiments.</p

HOIPΔRBR inhibits IκBα phosphorylation and degradation.

<p>TRAF6-deficient A20.2J cells were stably transfected with FLAG2X-tagged HOIP or HOIPΔRBR in an IPTG-inducible expression vector. A, Expression of FLAG2X-tagged HOIP or HOIPΔRBR in stably transfected cell lines. Western blots of cell lysates from uninduced and IPTG-induced cell lines were probed with anti-FLAG (left) and anti-HOIP (right). Blots were reprobed for actin to verify equivalent lane loading. B, The two cell lines were stimulated with CD154 (CD40 ligand) for the times indicated and then processed for Western blotting. Unstimulated cells (first lane) and cells incubated for 5 minutes with insect cells lacking CD154 served as negative controls. Western blotting for phosphorylated IκBα was performed; blots were then stripped and reprobed for total IκBα and actin. C, Quantification of IκBα degradation (panel B) in TRAF6-deficient A20.2J cells stably transfected with Lac repressor only (◊;♦), Lac repressor plus full-length HOIP (□;▪), or Lac repressor plus HOIPΔRBR (Δ; ▴). Filled symbols indicate cultures pre-treated with IPTG. IκBα bands in each lane were normalized to the actin signal. The degradation index is the fraction of IκBα remaining at each time point relative to the amount of IκBα present in cells incubated for 5 minutes with insect cells lacking CD154. The results presented are the mean of four experiments. Error bars indicate standard error of the mean.*, p<0.05 (one-sided Student's t test).</p

Western blot verification of mass spectrometry results.

<p>hmCD40 and hmCD40Δ67 ARC samples (3.3×10⁶ cell equivalents per lane) were fractionated by SDS-PAGE and transferred to PVDF membrane for Western blotting with the indicated antibodies. Lanes containing whole cell lysates (1.0×10⁵ cell equivalents per lane) were run in parallel. Each blot is representative of three or more experiments.</p

Proteins uniquely associated with ARC-purified hmCD40 as determined by mass spectrometry analysis.

<p>*All protein assignments listed met or exceeded the Scaffold 95% confidence filter.</p><p>**TRAF1 was detected in gel slices containing material at or above the known molecular weight for TRAF1 in hmCD40 ARC samples, but was also detected in both hmCD40Δ67 and hmCD40 gel slices containing material of approximately 35 kD.</p