Journal: Journal of Biological Chemistry

Article Title: dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

doi: 10.1074/jbc.m607530200

Figure Lengend Snippet: FIGURE 5. Dlk1 expression synergizes the effect of LPS-induced cytokines in hMSC cells and stimulates the B cell proliferation. A, control and hMSC-dlk1 cells were stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated and analyzed for the expression of the indicated genes by real time PCR. Expression of each target gene was normalized to -actin. Data are shown as means S.D. of three independent experiments. *, p 0.05; **, p 0.005 versus control cells (hMSC-TERT) induced with LPS. B, CD19-purified human peripheral blood B lymphocytes were cultured in CM obtained from hMSC-TERT (control cells) or hMSC-dlk1 cells as described under “Experimental Procedures” in the absence and the presence of 2.5 or 10 g/ml CD40L. Cell proliferation was measured with XTT-based assay. Stimulation in the cell proliferation was represented as fold induction over nonstimulated cells cultured in the corresponding CM. The bars are the means S.D. of five independent experiments. *, p 0.05; **, p 0.005 versus cultured cells in control/CM.

Article Snippet: Cells were then resuspended at 0.5 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated).

Techniques: Expressing, Control, Isolation, Real-time Polymerase Chain Reaction, Purification, Cell Culture, XTT Assay

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Control, Plasmid Preparation, Solvent

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Immunoprecipitation, Mutagenesis

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Elevated hepatic and serum ALCAM were observed in patients with AIH. (A) Representative immunohistochemistry images of ALCAM in liver biopsies from HC (n=3) and patients with AIH (n=6). (B) Representative immunofluorescence staining for CD4, CD6 and ALCAM in interface hepatitis lesion of liver sections from patients with AIH (n=3). (C) Concentration of serum ALCAM in HC (n=28) and AIH (n=86) was measured by ELISA assay. Individual correlation between clinical indicators and serum ALCAM was calculated in patients with AIH (n=86). (D) Correlation between the number of hepatic CD6 + cells and paired serum ALCAM concentration was calculated (n=27). ***p < 0.001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: ALCAM promoted CD6 high CD4 + T cells trans-endothelial migration in vitro . (A, B) Human CD4 + T cells were magnetically isolated from PBMC of healthy donors and stimulated with αCD3/28 for 3 days in a flat-bottom 96-well plate, the expression of CD6 and cell proliferation was detected with flow cytometry. (C) After stimulation, the expression of cytokines, surface markers and transcription factors was compared between the CD6 high and CD6 low subsets. (D) Pre-activated CD4 + T cells were placed on a transwell chamber with 5μm pore for 24 hours in the presence of rhALCAM (3ug/ml) or vehicle (PBS). The expression of CD6 and CD69 was measured by flow cytometry. Experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration, In Vitro, Isolation, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Schematic diagram for this study. Upregulated ALCAM on hepatocytes promoted the trans-endothelial migration of pathogenic CD6 high CD4 + T cells, which further aggravated hepatic inflammation of patients with AIH. This study revealed a putative therapeutic approach for patients with AIH.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Autophagy is induced in MT cells. ( A ) Mutant UQCRB-expressing cell lines were examined by Western blotting using anti-UQCRB and anti-Myc antibodies. ( B ) HEK293 and MT cells treated with either 10% serum-containing medium or serum-free medium for 24 h and then analyzed by Western blot analysis using antibodies against LC3B and p62. shows treatment of either 10% serum-containing medium or serum-free medium for 4 h. ( C ) Confocal microscopy images of HEK293 and MT cells treated with serum-free medium for 24 h after immunostaining with anti-LC3 antibody. Scale bar, 50 μm. ( D ) Western blot analysis results of LC3B and p62 levels in cells after treatment with 5 μM chloroquine (CQ) treatment for 0–6 h. ( E ) HEK293 and MT cells were treated with either deionized water (DIW) or CQ for 6 h. Cell extracts were subjected to Western blotting using LC3B and p62 antibodies. The densitometric value of LC3-II was quantitated based on Western blot data and normalized to LC3-I levels. ( F ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 for 24 h. Scale bar, 20 μm. ( G ) Numbers of mRFP-LC3 puncta were counted in HEK293 and MT cells ( n = 8). The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. Data are presented as the mean ± SD; statistical significance was assessed using an unpaired t -test. ns, p > 0.05.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Mutagenesis, Expressing, Western Blot, Confocal Microscopy, Immunostaining, Transfection, Control

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: TRPML1-induced lysosomal Ca 2+ release and TFEB activation are increased in MT cells. HEK293 and MT cells were transfected with GCaMP3-TRPML1 encoding a lysosome-specific Ca 2+ probe, 40 µM ML-SA1, for 120 s. ( A ) Confocal microscopy images of GCaMP green fluorescence showing HEK293 and ( B ) MT cells. ML-SA1 treatment for 120 s. ( C ) Confocal microscopy images of HEK293 and MT cells transfected with EGFP-TFEB and treated with the DMSO control or A1938 for 6 h. ( D ) The number of cells with a nuclear (Nuc) or cytoplasmic (Cyt) TFEB localization ratio is represented in the graph. Scale bar, 20 μm. Statistical significance was assessed using Student’s t -test. ** p < 0.01.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activation Assay, Transfection, Confocal Microscopy, Fluorescence, Control

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Lysosome activity in MT cells with or without A1938 treatment. ( A ) Confocal microscopy images and ( B ) numbers of acidic lysosome red puncta were counted in HEK293 and MT cells ( n = 15). The cells were treated with DMSO as a control or 30 µM A1938 for 24 h and then stained with LTR. Scale bar, 20 μm. ( C ) Confocal microscopy images of MT cells treated with DMSO as a control or 30 µM A1938 for 24 h, then the live cells were stained with 2 μg/mL acridine orange for 25 min and fixed. Scale bar, 20 μm. ( D ) Images of HEK293 and MT cell extracts treated with the DMSO control or 30 µM A1938 for 24 h in a serum-free medium, then subjected to Western blot analysis using antibodies against LAMP1. ( E ) Intensities of the LAMP1 immunoblot bands were normalized to β-actin expression. Western blot analysis out of three independent experiments. Statistical significance was assessed using Student’s t -test. ns, p > 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activity Assay, Confocal Microscopy, Control, Staining, Western Blot, Expressing

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: A1938 regulates autophagy flux by suppression of mROS generation in MT cells. ( A ) Cells treated with A1938 for 24 h in a serum-free medium. The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. ( B ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 and treated with DMSO, A1938, or Mito-Tempo (mTP) for 24 h. Scale bar, 20 μm. Quantification of data indicating red puncta (autolysosome) versus yellow puncta (autophagosome) is shown in ( C ). Values are the means ± SEM; statistical significance was assessed using a paired t -test. n = 10 cells, ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Control, Confocal Microscopy, Transfection

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) Domain structure of GDE2, GDE3 and GDE6 (left panel), and the transmembrane scheme of GDE3 (right panel). GDPD denotes catalytic glycerophosphodiesterase domain. Asterisk in GDPD domain depicts catalytic His residue in both GDE2 and GDE3. ( B ) Immunoblot analysis of uPAR release into the medium. HEK-uPAR cells transfected with empty vector (control), GDE2 or GDE3. PI-PLC served as positive control. ( C ) Mutant GDE3(H229A) fails to release uPAR. ( D ) Partial loss of uPAR from the plasma membrane by GDE3, as measured by flow cytometry. ( E ) TIRF microscopy reveals loss of uPAR from the basolateral plasma membrane. Box plot shows uPAR-GFP intensity at the ventral membrane (n = 3, mean ±SEM ****p<0001). ( F ) Homology modeling of the GDE2 and GDE3 catalytic domains showing surface charge distributions (blue, positive; red, negative; green line, putative GPI-binding groove; yellow line, proposed substrate-binding surface). The active site is indicated by glycerol-3-phosphate located at the template structure.

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Residue, Western Blot, Transfection, Plasmid Preparation, Control, Positive Control, Mutagenesis, Clinical Proteomics, Membrane, Flow Cytometry, Microscopy, Binding Assay

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) Confocal images of HEK293 cells expressing human GDE2-HA, GDE3-HA or GDE6-HA, as indicated; bar, 10 μm. ( B ) GDE3 localizes to distinct microdomains (yellow square, open arrows) and filopodia-like extensions (orange square, solid arrow), as visualized by confocal and super-resolution microscopy. Bars, 10 μm and 1 μm respectively. ( C ) Immunoblot analysis showing that GDE3 reduces the membrane-anchored uPAR pool and competes with PI-PLC. The medium and lysates of HEK-uPAR cells expressing GDE3 or empty vector (EV) were analyzed without or with PI-PLC treatment (45 min.) of the cells, as indicated (left and right panels, respectively). Right lanes refer to wild-type (WT) HEK293 cells. ( D ) Expression and localization of GDE3 and mutant GDE3(H229A), as measured by immunoblotting and confocal microscopy, respectively, at 24 hr after transfection. Actin was used as a loading control. ( E ) uPAR-TM containing the transmembrane domain of EGFR is not released by GDE3, as shown by immunoblotting using anti-uPAR antibody, while uPAR-TM is properly expressed at the plasma membrane (bar, 10 μm).

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Expressing, Super-Resolution Microscopy, Western Blot, Membrane, Plasmid Preparation, Mutagenesis, Confocal Microscopy, Transfection, Control, Clinical Proteomics

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) (Left) Scheme showing uPAR cleavage in cis or trans. (Right) GDE3-expressing HEK-uPAR cells were mixed with a GDE3-deficient cell population, as indicated. Immunoblot analysis of uPAR in medium and cell lysates indicates that GDE3 acts in cis; mock refers to empty vector-transfected cells. ( B ) GDE3 expression leads to increased GPI-free suPAR. Conditioned medium from HEK-uPAR cells expressing GDE2 or GDE3 was subjected to Triton X-114 phase separation. suPAR in the aqueous ( A ) and detergent ( D ) fractions was analyzed by immunoblotting. ( C ) GPI-anchor with phospholipase cleavage sites indicated; HONO, nitrous acid. ( D ) Representative LC-MS ion chromatograms (m/z 259.02–259.03); inositol 1-phosphate peaks in red. HONO-treated suPAR contains inositol 1-phosphate (n = 3, mean ±SEM; *p<0.05).

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) Endogenous uPAR expression in MDA-MB-231 versus HEK293 cells, as determined by immunoblot. ( B ) Endogenous GDPD2 expression, as determined by qPCR analysis. ( C ) (left) Cell-surface expression of GDE3-mCherry of MDA-MB-231 cells expressing GDE3, as detected by flow cytometry. (Right) Cell-surface expression of uPAR in control (grey) and GDE3-expressing MDA-MB-231 cells (red), as detected by flow cytometry. ( D ) Confocal (top) and dual-color super-resolution microscopy images (bottom) of MDA-MB-231 cells expressing GDE3-GFP or catalytically dead GDE3(H229A)-GFP. Endogenous uPAR was immunostained in red. Merged images show colocalization of uPAR with GDE3(H229A) but not with wild-type GDE3 and uPAR. Scale bars, 10 μm (confocal) and 1 μm (super-resolution). Co-localization analysis (Mander's coefficient) on peripheral uPAR patches in confocal images was done using ImageJ software (n = 30 cells, three independent experiments). ( E ) Endogenous uPAR staining in control, GDE3-overexpressing and GDE3 knockout MDA-MB-231 cells plated on vitronectin. Two distinct GDE3 knockout clones (KO1 and KO2) were examined, as indicated. Scale bar,10 μm. ( F ) Quantification of basolateral uPAR-containing membrane domains referring to the cells in panel ( E ) (n = 3, mean ±SEM, ****p<0.0001). GDE3 suppresses the vitronectin- and uPAR-dependent phenotype of MDA-MB-231 breast cancer cells.

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Expressing, Western Blot, Flow Cytometry, Control, Super-Resolution Microscopy, Software, Staining, Knock-Out, Clone Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 1. TAK1 regulates Nod2-mediated NF-B activation. A, the dominant negative form of TAK1 (TAK1DN) inhibits NF-B activation induced by Nod2. HEK293T cells were transfected with pCDNA-Nod2 (3 ng) and reporter constructs RSV-KB-Luc and RSV--galactosidase plus the dominant negative forms of TBK1, Ubc13, Rip, or TAK1 (120 ng each) or control vector. Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated at least three times with equivalent results. *, p 0.0001 (vector versus TAK1DN). B, the dose effect of TAK1DN on Nod2-mediated NF-B. HEK293T cells were transfected with fixed amounts of pCDNA-Nod2 (3 ng), RSV-KB- Luc, and RSV--galactosidase plus the indicated doses of TAK1DN. The luciferase activity of cells transfected with reporters and Nod2 alone (control) was arbitrarily set at 100, and the percent of inhibition was calculated accordingly. The experiments were performed in triplicate and repeated at least three times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Dominant Negative Mutation, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Activity Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 2. Dominant negative form of TAK1 (TAK1DN) inhibits MDP-induced NF-B activation in Nod2-expressing cells. HEK293T cells were transfected with pCDNA-Nod2 (5 ng) and reporter constructs plus the indicated amounts of TAK1DN or control vector. Cells were stimulated with MDP (100 ng/ml) 8 h after transfection. Luciferase activity was determined 16 h after stimulation and normal- ized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent re- sults. *, p 0.05.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Dominant Negative Mutation, Activation Assay, Expressing, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 3. Nod2 interacts with TAK1. A, Nod2 coimmunoprecipitates with TAK1. HEK293T cells were cotransfected with plasmid pCMV-HA- TAK1 (4 g) plus 4 g of DNA pLPCX-FLAG-Nod2, control vector pLPCX-FLAG, or pLPCX-FLAG-LRR. Immunoprecipitations (IP) were performed with anti-HA antibodies and subjected to Western blot (IB) analysis using anti-FLAG antibodies to detect FLAG-Nod2 or FLAG-LRR (left). Immunoprecipitations were also performed using anti-FLAG antibodies and subjected to Western blot analysis using anti-HA antibodies to detect TAK1 (right). All experiments were repeated three times with equivalent results. B, coprecipitation of endogenous TAK1 and Nod2. LS174T and THP-1 cell lysates were prepared as described under “Experimental Procedures.” Immunoprecipitations were performed with anti-Nod2 antibodies or control Ig and subjected to Western blot analysis using anti-TAK1 antibodies (top panel). The membranes were reprobed with anti-Nod2 (middle panel). Aliquots of cell lysates were immunoblotted with anti-TAK1 (bottom panel). C, the LRR region of Nod2 interacts with TAK1. Full-length TAK1 translated in vitro and labeled with 35S was incubated with GSTLRR fusion protein, GSTCARD, or GST protein. Input TAK1 indicates 1/10 of 35S-labeled protein used in each GST pull-down assay. All experiments were repeated two times with equivalent results. D, the N-terminal of TAK1 interacts with the LRR region of Nod2. Full-length (FL) and truncated forms of TAK1 (aa 1–303, 1–403, and 286–632) were generated by in vitro transcription and translation and incubated with GSTLRR or GST protein. Input TAK1 indicates 1/10 of 35S-labeled protein used in each GST pull-down assay. All experiments were repeated three times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Plasmid Preparation, Control, Western Blot, In Vitro, Labeling, Incubation, Pull Down Assay, Generated

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 5. Nod2 does not induce TAK1 activation. HEK293T cells were transfected with equal amounts of the indicated expression con- structs: control vector and TAK1 (leftmost lane), Nod2 and HA-TAK1 (3 g) (middle lane), TAB1 and TAK1 (rightmost lane). TAK1 protein was immunoprecipitated with anti-TAK1 antibody and subjected to phos- phorylation as described under “Experimental Procedures.” All experi- ments were repeated two times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Transfection, Expressing, Control, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 6. Coexpression of Nod2 and TAK1 does not exert a syn- ergistic effect on NF-B activation. HEK293T cells were trans- fected with Nod2 (5 ng) alone, TAK1 (25 ng), and TAB1 (2 ng) together, TAK1 (25 ng) and TAB1 (2 ng) plus Nod2 (5 ng), or TAK1 (25 ng) and TAB1 (2 ng) plus control vector (5 ng). Expression plasmids RSV-KB- Luc and RSV--galactosidase were included in each transfection. Lu- ciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and were repeated three times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Control, Plasmid Preparation, Expressing, Transfection, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 7. Nod2 suppresses TAK1-induced NF-B activation. A, TAK1 induces NF-B activation in RICK/ fibroblasts. RICK/ fibroblasts were seeded in 12-well plates (105/well). Reporter constructs with TAK1 (25 ng) and TAB1 (2 ng) and without (none) were cotransfected into cells by FuGENE 6. B, TAK1 activates NF-B equally well in both RICK/ and wild-type fibroblasts. RICK wild type (wild, shaded bar) and Rick/ (null, solid bar) fibroblasts were transfected with the indicated amounts of TAK1 and TAB1, along with the reporter constructs. Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated two times with equivalent results. C, Nod2 inhibits TAK1-induced NF-B activation in RICK/ fibroblasts. RICK/ fibroblasts were transfected with Nod2 alone (LPCX-Nod2, 50 ng) or with TAK1 (25 ng) and TAB1 (2 ng) plus the indicated amounts of Nod2 (LPCX-Nod2, 0, 25, 50 ng) or control vector (LPCX, 25, 50 ng). Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. *, p 0.01 (Nod2 versus control vector). D, Nod2 siRNA efficiently blocks expression of transfected Nod2. HEK293T cells were transiently transfected with Nod2-specific siRNA or control siRNA, and 24 h later, they were transfected with FLAG-Nod2. Cell lysates were prepared 24 h after the Nod2 transfection and subjected to SDS-PAGE and immunoblotting by anti-FLAG antibody. The arrows indicate the Nod2 and nonspecific (NS) bands. E, expression of Nod2 siRNA suppresses endogenous NF-B activity. LS174T cells were transfected with either Nod2-specific siRNA or control siRNA plus the reporter constructs. Luciferase activity was determined 24 h after transfection as in C. All experiments were performed in triplicate and repeated three times with equivalent results. *, p 0.05. F, expression of Nod2 siRNA potentiates TAK1-induced NF-B activation. LS174T cells were transfected with Nod2-specific siRNA or control siRNA. These cells were then transfected with TAK1 (25 ng), TAB1 (2 ng), and the reporter constructs 24 h after the siRNA transfection. Luciferase activity was analyzed as in C. The results represent the mean of two independent experiments. Increase in NF-B activation in the control siRNA group was arbitrarily set at 1.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Construct, Transfection, Luciferase, Activity Assay, Control, Plasmid Preparation, Expressing, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 8. Inhibitory effect of the LRR region on TAK1-induced NF-B acti- vation. A, the LRR region of Nod2 sup- presses TAK1-induced NF-B activation. HEK293T cells were transfected with TAK1 (25 ng) and TAB1 (2 ng) plus LRR in the amounts indicated. HEK293T cells transfected with identical amounts of TAK1 and TAB1 and control vector were used as the control. Luciferase activity was determined 24 h after transfection and normalized on the basis of -galacto- sidase activity. All experiments were per- formed in triplicate and repeated three times with equivalent results. B, mutant LRR is less effective than wild-type LRR at suppressing TAK1-mediated NF-B ac- tivation. HEK293T cells were transfected with TAK1 (25 ng) and TAB1 (2 ng) plus wild-type or mutant LRR in the amounts indicated (left). Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. *, p 0.05 (LRR versus mutant LRR). A cell lysate fraction used for the luciferase assay was sub- jected to Western blot analysis to deter- mine the expression of TAK1, TAB1, and LRR, both wild type and mutant (right).

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Western Blot, Expressing