soluble gst fusion  (GE Healthcare)

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: Nucleic Acids Research

Article Title: Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum

doi: 10.1093/nar/gkz1044

Figure Lengend Snippet: The HAT module of a SAGA-like complex is recruited to H3K4me2/me3 via an unconventional PhD-finger containing reader. ( A ) Scatterplots of log 2 normalized ratios of protein enrichments in histone peptide pull downs: H3K4me1, me2 and me3 over unmodified peptide corresponding to the 21 amino acid N-terminal sequence of P. falciparum H3.3. Scatterplots with auto-scaled axes and heatmaps summarizing all H3K4me pulldown experiments can be found in . Components of the GCN5/ADA2 core complex are highlighted (note that their enrichment on the H3K4me1 pull down is not significant). ( B ) The domain structure of PHD1 (PF3D7_1008100) as identified by the SMART algorithm using default settings (top panel). Alignment of the PHD-domain sequences of PHD1 against human CHD4 PHD domain. Key features of PHD domains are highlighted on the alignment: conserved zinc-coordinating residues are labelled grey; two core β-strands are indicated by green arrows; regions involved in ligand recognition and selectivity are numbered I–V, with residues known to be important for H3K4me3 recognition highlighted blue . ( C ) Western blot quantifying the amount of recombinant glutathione S-transferase (GST) protein fused to the fourth PhD-finger of PHD1 before (10% input) or after pull-down with unmodified, K4me1, K4me2 or K4me3 H3.3 N-terminal peptides. The composition of each pull-down reaction is indicated below the image (+ present; - absent). ( D ) Heatmaps depicting the log2 normalized GFP/HA-over-control ratios as well as the false discovery rates (FDR) for proteins identified in co-immunoprecipitation experiments using parasite lines in which GCN5, PHD1 or PHD2 was endogenously tagged with GFP or 3xHA as indicated. Significant outliers were identified for each reaction by means of intensity-based outlier statistics (two-sided Benjamini–Hochberg test). Proteins significantly enriched with an FDR < 10% in at least two reactions for GCN5, PHD1 and/or PHD2 were selected, while excluding proteins significantly enriched with the same criteria in the GFP- and/or HA-negative controls using wild-type nuclear extracts. Columns were clustered using a sequential hierarchical clustering approach. Rows were ordered manually, first listing the proteins strongly recruited by PHD1 and then ranking based on the strength of recruitment in the combined GCN5 pulldown from high to low summed log 2 -norm ratios.

Article Snippet: Soluble GST-fusion protein was purified by glutathione sepharose 4b (GE Healthcare) based on the manufacture's protocol.

Techniques: Sequencing, Western Blot, Recombinant, Immunoprecipitation

Journal: Oncotarget

Article Title: Melphalan modifies the bone microenvironment by enhancing osteoclast formation

doi: 10.18632/oncotarget.19152

Figure Lengend Snippet: (A-C) Flow cytometry analysis of bone marrow cells from mice treated with vehicle (Veh.) or melphalan (Mel.) for 3 days. (A) Total live cells were analyzed as a proportion of all cells. Live cells were then analyzed to determine relative numbers of (B) monocytes/macrophages (CD115 + ), (C) immature macrophage subpopulations (CD11b − /GR1 − /CD115 + ) and more mature macrophages (CD11b + /Gr1 − /CD115 + ). (D) Bone marrow cells from mice treated with vehicle or melphalan were assessed for osteoclast formation ex vivo in the presence of 25ng/ml M-CSF in combination with 0, 10 and 20ng/ml RANKL for 6 days; scale bar = 50μm. For graphs in (B) and (C) data is presented as individual data points with indicated mean ± SD. *p<0.05; **p<0.01; ***p<0.001 using Student's t test (n = 8). Graph in (D) shows mean and S.E.M. using ANOVA/Dunnett's post hoc test (n = 8).

Article Snippet: Recombinant murine soluble RANKL (RANKL 158-316 –GST fusion protein) was obtained from the Oriental Yeast Co. (Tokyo, Japan) and human M-CSF from Sino Biological Inc. (Beijing, P.R.

Techniques: Flow Cytometry, Ex Vivo

Journal: Oncotarget

Article Title: Melphalan modifies the bone microenvironment by enhancing osteoclast formation

doi: 10.18632/oncotarget.19152

Figure Lengend Snippet: (A) Histochemical analysis of TRAP + multinucleated cells (MNCs) of bone marrow cells cultured in 20ng/ml RANKL and 25ng/ml M-CSF in combination with doses of melphalan or vehicle for 6 days. (i) Photomicrographs showing histochemical staining of TRAP + MNCs indicated by arrowheads (boxed area shown at higher magnification in inset image) and (ii) quantitative dose response data from cultures (n = 4). Scale bars = 50 μm. (B) Histochemical analysis of TRAP + MNCs of RAW264.7 cells cultured in 20ng/ml RANKL with melphalan or vehicle for 6 days (i) Photomicrographs showing histochemical staining of TRAP + MNCs indicated by arrowheads and (ii) quantitative dose response data from cultures (n = 4). Scale bars = 50 μm. (C) Simplified diagram of transcription factor induction cascade induced by RANKL/RANK binding causing increased levels of active NFkB, NFATc1 and MITF. Note many important factors in this cascade (e.g., AP-1 and p38) are omitted for clarity. RANKL and melphalan treatment for 24 h on the induction of signals in (D) NFkB-RAW and (E) NFAT-RAW reporter cells, showing induction by RANKL but not by melphalan. (F) Western blot analysis of NFATc1 protein levels in RAW264.7 cells treated with RANKL, vehicle or melphalan for 48 h. (G) Western blot analysis of MITF protein levels of bone marrow macrophages (BMMs) and RAW264.7 cells treated with vehicle or melphalan in the absence of RANKL for 48 h. (H) qRT-PCR-analysis of osteoclast-associated mRNA levels in RAW264.7 cells treated with vehicle or melphalan in the absence of RANKL for 24 h. (I) Histochemical analysis of MNCs formed in RAW264.7 cells treated with vehicle or melphalan in the absence of RANKL for 6 days. (i) Photomicrographs of MNCs as indicated by arrowheads and (ii) quantification of the MNCs. Scale bars = 50μm. (J) Quantification of TRAP + MNCs in RAW264.7 cells treated with 2μM melphalan in the presence of RANKL for 6 days. In photomicrographs (A, B and I) the boxed areas shown at higher magnification in inset images. Quantitative data from in vitro experiments presented as mean ± S.E.M. from 4 independent experiments. *p<0.05, **p<0.01, ***p<0.001, using ANOVA/Dunnett's post hoc test.

Article Snippet: Recombinant murine soluble RANKL (RANKL 158-316 –GST fusion protein) was obtained from the Oriental Yeast Co. (Tokyo, Japan) and human M-CSF from Sino Biological Inc. (Beijing, P.R.

Techniques: Cell Culture, Staining, Binding Assay, Western Blot, Quantitative RT-PCR, In Vitro

Journal: Oncotarget

Article Title: Melphalan modifies the bone microenvironment by enhancing osteoclast formation

doi: 10.18632/oncotarget.19152

Figure Lengend Snippet: (A) Western blot analysis of Hsp70 protein levels in RAW264.7 cells treated with melphalan in the absence and presence of HSP inhibitor KNK437 for 48 h. (B) Histochemical analysis of TRAP + MNCs in RAW264.7 cells treated with RANKL and melphalan in the absence and presence of KNK437 for 6 days. Scale bars = 50 μm (i) Photomicrographs of TRAP + MNCs as indicated by arrowheads and (ii) quantitative data showing the effects of KNK437 on TRAP + MNCs formed in RANKL and melphalan and RANKL treated cultures of RAW264.7 cells. (C) Histochemical analysis of TRAP + MNCs in bone marrow cells treated with M-CSF, RANKL and melphalan in the absence and presence of KNK437 for 6 days. (i) Photomicrographs of TRAP + MNCs as indicated by arrowheads and (ii) quantitative data showing the effects of KNK437 on TRAP + MNCs formed in RANKL and melphalan treated cultures. In photomicrographs (B and C) the boxed areas shown at higher magnification in inset images. Scale bars = 50 μm. Data presented as mean ±S.E.M. from 4 independent experiments. *p<0.05, **p<0.01, ***p<0.001 relative to vehicle treated controls, using ANOVA/Dunnett's post hoc test.

Article Snippet: Recombinant murine soluble RANKL (RANKL 158-316 –GST fusion protein) was obtained from the Oriental Yeast Co. (Tokyo, Japan) and human M-CSF from Sino Biological Inc. (Beijing, P.R.

Techniques: Western Blot

Journal: Oncotarget

Article Title: Melphalan modifies the bone microenvironment by enhancing osteoclast formation

doi: 10.18632/oncotarget.19152

Figure Lengend Snippet: qRT-PCR analysis of RANKL and OPG mRNA in BMSC treated with (A) 1,25-D3 or (B) melphalan for 24 h (n = 3). (C) Histochemical analysis of TRAP + MNCs in co-cultures of fresh bone marrow cells and in vitro expanded BMSCs treated with vehicle, 1,25-D3 or melphalan for 6 days (n = 3). Scale bars = 50μm. (D) RANKL and OPG mRNA levels were determined in whole bones (tibiae) of mice treated for 3 days with melphalan or vehicle (n = 8). Data presented as mean ±S.E.M. *p<0.05, **p<0.01, ***p<0.001 relative to vehicle treated controls, using ANOVA/Dunnett's post hoc test or t-test for 2-way comparison.

Article Snippet: Recombinant murine soluble RANKL (RANKL 158-316 –GST fusion protein) was obtained from the Oriental Yeast Co. (Tokyo, Japan) and human M-CSF from Sino Biological Inc. (Beijing, P.R.

Techniques: Quantitative RT-PCR, In Vitro, Comparison

Journal: Oncotarget

Article Title: Melphalan modifies the bone microenvironment by enhancing osteoclast formation

doi: 10.18632/oncotarget.19152

Figure Lengend Snippet: Melphalan slightly increased RANKL expression in osteoblastic stromal cells (dotted line); enhanced osteoclast progenitor numbers in vivo ; increased differentiation in vitro at least partly in a cell stress dependent manner; enhanced MITF levels in osteoclast progenitors; and increased cell fusion possibly through increased Dc- and Oc-stamp levels. Increased osteoclast numbers seen both in vitro and in vivo with mephalan treatment can result in increased bone resorption and thus may potentially also enhance growth of tumor cells or activation of dormant tumors.

Article Snippet: Recombinant murine soluble RANKL (RANKL 158-316 –GST fusion protein) was obtained from the Oriental Yeast Co. (Tokyo, Japan) and human M-CSF from Sino Biological Inc. (Beijing, P.R.

Techniques: Expressing, In Vivo, In Vitro, Activation Assay