Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD4 + CD28 null T cell clones express CD158b/j, but do not express KARAP/DAP12. CD4 + CD28 null T cells were sorted from patients with RA, and clones were established by limiting dilution. Clones were analyzed by flow cytometry for expression of CD28 and CD158b/j. Four representative clones (#1 through #4) are shown. All clones expressed CD4 (unpublished data; A). RT-PCR was used to amplify transcripts for KARAP/DAP12 and β-actin from PBMCs (lane 1), Jurkat T cells (lane 2), and CD4 + CD28 null T cell clones #1–#4 (lanes 3–6, respectively). cDNA was omitted for the negative control (lane 7) (B). Western blotting was used to detect KARAP/DAP12 and β-actin protein (bottom panels) in Jurkat T cells (lane 1), Jurkat T cells transfected with KARAP/DAP12 + vaccinia virus (lane 2), and CD4 + CD28 null T cell clones (lanes 3–7) (C).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Clone Assay, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Transfection, Virus

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Stimulation through CD158b/j results in an up-regulation of ATF-2 and HSP27 transcripts. The PathwayFinder cDNA Array is spotted in duplicate with 23 cDNAs. Represented on the membrane are the ERK (egr-1 and c-fos), JNK (ATF-2, hsf1, HSP27, and HSP90), NF-κB (iNos, NF-κB, and IκBα), NFAT (IL-2, Fas, and CD5), TGF-β (p16, p21, and p57 Kip2 ), Wnt (c-myc), p53 (p21, gadd45, pig7, pig8, mdm2, and bax), and CREB pathways (egr-1, CYP19, and c-fos). The membrane also included a negative control (pUC18) and two positive controls (β-actin and GAPDH) (A). A CD4 + CD28 null CD158b/j + T cell clone was stimulated with control mouse IgG or anti-CD158j mAb and cross-linked with rabbit anti–mouse IgG Ab. Total RNA was harvested and used to probe the PathwayFinder cDNA Array (B).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Membrane, Negative Control, Control

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Phosphorylation of JNK is initiated by stimulation specifically through CD158j. Two CD4 + CD28 null CD158j + T cell clones (top panels) and a CD4 + CD28 null CD158b1 + T cell clone (bottom panels) were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels; A). Jurkat T cells were infected with either wild-type vaccinia virus (WR) or vaccinia virus containing CD158j cDNA and were analyzed for expression of CD158j by flow cytometry (B). Jurkat T cells infected with WR vaccinia virus or CD158j + vaccinia virus were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (top left panels) and MKK4 (bottom left panel). The blots were stripped and reprobed with Abs against β-actin (top right panels) or MKK4 (bottom right panel) (C).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Phospho-proteomics, Clone Assay, SDS Page, Membrane, Infection, Virus, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Mutation of transmembrane lysine residue in CD158j abolishes ability to induce JNK phosphorylation. Jurkat T cells were transiently transfected with constructs containing the CD158j cDNA or the CD158j233I cDNA. Cell-surface expression was confirmed by flow cytometry (A). Jurkat T cells transfected with either CD158j or CD158jK233I were stimulated with anti-CD3 or anti-CD158b/j mAb and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels) (B).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Mutagenesis, Residue, Phospho-proteomics, Transfection, Construct, Expressing, Flow Cytometry, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by streptavidin-HRP (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Negative Control, SDS Page, Membrane, Phospho-proteomics, Expressing, Stable Transfection, Transfection, Flow Cytometry, Immunoprecipitation, Western Blot