Journal: Journal of Biological Chemistry

Article Title: Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

doi: 10.1074/jbc.m309338200

Figure Lengend Snippet: FIG. 6. Functional characterization of AtOPT3. A, hybridization of an 600-bp AtOPT3 cDNA probe, corresponding to the 3-terminal exon, to RNA extracted from Arabidopsis roots of plants grown in control conditions (Ctl), copper (-Cu), manganese (-Mn), zinc (-Zn), and iron (-Fe) deficiency. The ethidium bromide-stained RNA gel is shown for quantification. B, growth of ctr1 expressing AtOPT3 on YPG-Ura plates supplemented with 10 M CuSO4 compared with the mutant transformed with the vector alone. C, growth of smf1 expressing AtOPT3 on manganese-limited medium, with and without 1 mM EGTA compared with the growth of the mutant transformed with the vector alone.

Article Snippet: Genome-wide Analysis Provides Insight into Metal Transport—We have used Affymetrix Arabidopsis DNA chips containing 8,300 genes (which cover about one-third of the ge- FIG. 7.

Techniques: Functional Assay, Hybridization, Control, Staining, Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA . ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Reverse Transcription, Purification, Amplification, In Vitro

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Experimental procedure utilized for the analysis of 10-year-old matched frozen and formalin-fixed paraffin embedded breast cancer samples. ( a ) Five micrograms of RNA extracted from the 10-year-old frozen portion of the sample, is reverse-transcribed and the cDNA is double stranded (dsDNA), in four individual reactions. The dsDNA of three reactions undergoes IVT-amplification (MessageAmpII, Ambion), which gives rise to complementary RNA (cRNA) for cDNA microarray analyses. The dsDNA of one reaction is used for PCR experiments. ( b ) Five micrograms of RNA extracted from the 10-year-old FFPE portion of the sample underwent the exact same process. ( c ) Single-stranded DNA (ssDNA) obtained by RT of 5 μg of FFPE-RNA is purified and hybridized to the sense-RNA template library. The restored ssDNA is double stranded and purified. Three of the CT-RT reactions undergo IVT-amplification, while the dsDNA of one reaction is used for PCR experiments.

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Formalin-fixed Paraffin-Embedded, Reverse Transcription, Amplification, Microarray, Purification

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Signal intensity and heat-map analysis of the correlation between the log 2 ratios measured by cDNA microarrays. ( a ) Signal intensity of one sample grid in the red channel (Cy5) across all microarrays. Top three panels display the grids obtained from three repeats using cRNA from 10-year-old frozen RNA (Frozen-Amp 1–3). Three mid-panels show the signal of three repeats using cRNA obtained by restoration and IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Restored 1–3). Three bottom panels display the signal of three repeats using cRNA obtained by direct IVT-amplification of RNA from 10-year-old FFPE tissue. ( b ) Heat map displaying the log 2 of expression ratios ranging between 0.5 and 2 for 1044 genes detected in frozen tissue on a 28 032 features cDNA microarray and represented in the UHR library. From left to right are displayed the ratios obtained by IVT-amplification of RNA from 10-year-old frozen tissue (Frozen-Amp 1–3), restoration and IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Restored 1–3) and direct IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Amp 1–3). Each column represents an individual hybridization and each line a different feature. Red and blue represent up-regulated and down-regulated genes, respectively.

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Amplification, Expressing, Microarray, Hybridization

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