Journal: PLoS ONE

Article Title: Expanding the Substantial Interactome of NEMO Using Protein Microarrays

doi: 10.1371/journal.pone.0008799

Figure Lengend Snippet: (A) Domain structure of the human NEMO protein, showing the two coiled coil domains (CC1 and CC2), the NEMO ubiquitin binding domain (NUB), leucine zipper (LZ) and zinc finger (ZF). (B) Immunoblot detection of biotinylated NEMO following purification of GST-NEMO, cleavage of the GST tag and biotinylation. Biotinylated NEMO was detected with streptavidin-alkaline phosphatase conjugate. (C) Example hits obtained from the array, compared to the same spot positions on negative control array. (D) Frequency histogram for the NEMO-probed protein microarray showing the range of Z-scores obtained. Protein interactors with a Z -score greater than three (Z>3; P<0.002) were deemed significant. Scores obtained for the canonical NEMO interactors IKKalpha (Z = 6.52) and IKKbeta (Z = 8.41) are shown for reference. Scores were calculated using Invitrogen Protoarray Prospector version 5.1 software. See for gene descriptions.

Article Snippet: Human NEMO was expressed as a soluble GST fusion from the pGEX-4T vector in E. coli BL21(DE3) and purified using glutathione sepharose (GE Life Sciences) as described previously .

Techniques: Binding Assay, Western Blot, Purification, Negative Control, Microarray, Software

Journal: PLoS ONE

Article Title: Expanding the Substantial Interactome of NEMO Using Protein Microarrays

doi: 10.1371/journal.pone.0008799

Figure Lengend Snippet: (A) Immunoblot analysis of GST and GST-NEMO proteins used as control and bait for the pulldown assay. Proteins were detected using anti-GST/HRP conjugate following SDS-PAGE and membrane transfer. (B) Results of GST pulldown assays showing binding of NEMO to putative interactors identified by protein array screening. Each of the interactors and IKKbeta, a known NEMO binder, were overexpressed in transiently transfected HEK-293T cells and the resulting lysates applied to immobilized GST or GST-NEMO. Following incubation and washing, the samples were resolved by SDS-PAGE and the proteins detected using appropriate antibodies. Input lanes were loaded with 5–10% of HEK-293T lysates to confirm protein expression. The size of relevant protein markers is shown beside the blot image. (C–H) Coimmunoprecipitation assays between NEMO and putative binders in HEK-293T cells. Plasmids encoding Xpress-tagged NEMO or the empty parent vector and tagged putative binders were used to transfect HEK-293T cells and the resulting cell lysates used for coimmunoprecipitation assays. For each putative binder, immunoblots are shown for detection of the binder using a tag- or protein-specific antibody, and for detection of Xpress-tagged NEMO. For IKKbeta and each of the five putative interactors, substantial coimmunoprecipitation occurred only in the presence immunoprecipitated NEMO. Input lanes contained 5–10% of the precleared input volume used prior to addition of anti-Xpress antibody. Binding and washing steps were performed in the presence of 0.5% NP-40 for all proteins except SAG, where 0.1% NP-40 was used. (I) NEMO interacts with CALB1, CDK2, SAG, SENP2 and SYT1 in a mammalian two-hybrid system. Empty two-hybrid vectors were cotransfected as a negative control. The MyoD/Id and NEMO/IkappaBalpha protein pairs were used as positive controls, while putative interaction partners cotransfected with empty complementing vector were used as negative controls. For each pair tested, a significant increase (n = 6; two-tailed T test; P≤0.05) in luciferase activity was obtained in partner/NEMO experiments compared to partner/vector experiments (indicated by asterisks).

Article Snippet: Human NEMO was expressed as a soluble GST fusion from the pGEX-4T vector in E. coli BL21(DE3) and purified using glutathione sepharose (GE Life Sciences) as described previously .

Techniques: Western Blot, SDS Page, Binding Assay, Protein Array, Transfection, Incubation, Expressing, Plasmid Preparation, Immunoprecipitation, Negative Control, Two Tailed Test, Luciferase, Activity Assay

Journal: Respiratory Research

Article Title: Expression profiling identifies genes involved in emphysema severity

doi: 10.1186/1465-9921-10-81

Figure Lengend Snippet: Comparison of mRNA expression in seven candidate genes between TPCH test (n = 30, microarray) and training set (n = 62 qRT-PCR data), with two public microarray datasets of lung tissue samples (Spira et al , , n = 34; and Golpon et al , n = 10) . Fold change represents mean expression ratio of moderate versus mild emphysema (TPCH training set), severe/mild emphysema versus normal (Spira et al ), or severe emphysema versus normal samples (Golpon et al ). The absence of a bar indicates the gene was not represented on the microarray platform.

Article Snippet: Lung and universal reference RNA (Stratagene, La Jolla, CA, USA) was reverse transcribed, labeled with Cy5 and Cy3 (Amersham/GE Healthcare, Buckinghamshire, England) respectively and co-hybridized onto a 22K Operon V2.1 Human Genome Oligo Microarray chip http://www.operon.com containing 21,329 70 mer probes representing ~14,200 named transcripts printed by the British Columbia Gene Array Facility http://www.microarray.prostatecentre.com .

Techniques: Expressing, Microarray, Quantitative RT-PCR