Journal: Oncogene

Article Title: Inhibition of c-Met Downregulates TIGAR Expression and Reduces NADPH Production Leading to Cell Death

doi: 10.1038/onc.2010.490

Figure Lengend Snippet: (A) Apoptosis of NPC cells is associated with p53 and TIGAR downregulation. Cell death by apoptosis (as measured by DNA fragmentation) was quantitatively determined using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics, Germany) according to the manufacturer’s instruction. Cells were treated with AM7 (2μM) or SU11274 (5μM) or DMSO for 48h and harvested for cell death quantification. Fold change in apoptosis relative to the respective DMSO control was presented for each cell line (mean ± SEM, n=3) and similar results were obtained in 3 independent experiments. Lower panel: Western blots showing the expression levels of TIGAR and p53 in HK1-LMP1 and CNE-2 cells at 48h of AM7 (2μM) or SU11274 (5μM) or DMSO treatment (See Supplementary Information for antibody sources). Similar results were obtained in 3 independent experiments. (B). AM7 induces dose-dependent reduction of intracellular NADPH in NPC cells. HK1-LMP1 and CNE-2 cells were treated with AM7 (2μM) or DMSO in complete medium for 48h. Cellular NADPH production was measured at 30min according to the manufacturer’s instruction (See details in Supplementary Information) and normalized to total protein as μM/min/mg total protein. Percentage reduction of NADPH was calculated with reference to DMSO. Graph show percentage reduction of NADPH level of AM7-treated cells vs DMSO-treated cells (mean ± SEM, n=3). Similar results were obtained in 3 independent experiments. (C) A model c-Met TKI, SU11274 downregulates TIGAR and NADPH levels in NPC cells. HK1-LMP1 and CNE-2 cells were treated with SU11274 or DMSO in complete medium for 48h. Cellular NADPH production was determined as above. The expression levels of TIGAR and p53 were also shown. (D) Overexpression of TIGAR rescues NPC cells from both AM7- and SU11274-mediated growth inhibition. Infective retroviruses expressing human TIGAR gene (original gene construct of human TIGAR was obtained from Origene, USA) or the control vector (pLPCX control vector for retroviral gene delivery; Clontech, USA) were generated. Retrovirus infection into NPC cells (CNE-2 and HONE-1) was performed, followed by puromycin selection (400ng/ml) as previously described (Lui et al., 2009b). Upon confirmation of TIGAR overexpression (by Western blotting for TIGAR; Upper Panel) in these stable cell lines, puromycin selection was removed periodically. Retrovirus-infected stable cell lines from CNE-2 and HONE-1 origin were treated with AM7 (2μM) or SU11274 (5μM) or DMSO in 3% FBS for 72h. Effects of TIGAR overexpression on AM7- or SU11274-induced growth inhibition (as % growth inhibition vs DMSO control) was assayed by MTT assay and presented in the middle and lower panels, respectively. Similar results were obtained in 3 independent experiments.

Article Snippet: Using retrovirus infection, we generated stable cell lines expressing TIGAR protein (human TIGAR gene construct was purchased from Origene, USA) ( ).

Techniques: Control, Western Blot, Expressing, Over Expression, Inhibition, Construct, Plasmid Preparation, Retroviral, Generated, Infection, Selection, Stable Transfection, MTT Assay

Journal: Redox Biology

Article Title: The E3 ubiquitin ligase TRIM31 is involved in cerebral ischemic injury by promoting degradation of TIGAR

doi: 10.1016/j.redox.2021.102058

Figure Lengend Snippet: TRIM31 targeted TIGAR for its polyubiquitination and proteasomal degradation. (A) PC12 cells were transfected with GFP-TRIM31 expression plasmid or its mock control. The transfected cells were cultured for 24 h before being further incubated with cyclohexamide (CHX) for the indicated time. The levels of TIGAR at different time points were detected by Western blot. (B) TIGAR protein levels were quantitated by measuring band intensities and normalized to GAPDH. (C) Western blot analysis of extracts from HEK293T cells transfected with Flag-TIGAR and GFP-TRIM31 expression plasmids then treated with MG132 (10 mM), chloroquine (10 mM) or 3-MA (10 mM) for 4 h. (D) Co-IP analysis of the exogenous interaction of TRIM31 with TIGAR, transfected with plasmids expressing GFP-TRIM31 and Flag-TIGAR in HEK 293T cells. (E) CO-IP analysis of the endogenous interaction of TRIM31 with TIGAR in PC12 cells after the treatment of OGD/R. (F) Representative images of laser scanning confocal microscopy for TRIM31 (red) and TIGAR (green) in primary neuron cells. Scale bars: 10 μm. (G) Schematic diagram of TRIM31 and its truncation mutants. (H) Flag-tagged TRIM31 or its mutants and Myc-TIGAR were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (I) Co-IP analysis of the ubiquitination of TIGAR transfected with plasmids expressing Myc-TIGAR, TRIM31 WT or C53A, C56A, and HA-ubiquitin in HEK293T cells. Presented figures are representative data from 3 independent experiments *P < 0.05, for the statistical analysis of the indicated groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies against TIGAR (sc‐166 290), Tubulin (sc‐80005) were from Santa Cruz (Dallas, TX, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Cell Culture, Incubation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Immunoprecipitation, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis

doi: 10.1038/s41467-024-48708-0

Figure Lengend Snippet: a HEK293 cells were transfected by His-TAK1, Flag-TIGAR, or Flag empty vector. Co-IP and western blot of TAK1 and TIGAR in the transfected cells. b HEK293 cells were transfected by Flag-TIGAR, HA-TAK1, or HA empty vector. Co-IP and western blot of TIGAR and TAK1 in the transfected cells. c Co-IP and western blot of endogenous TIGAR and TAK1 in BMDMs. d Co-IP and quantifications of endogenous TIGAR and TAK1 in BMDMs stimulated by LPS. n = 3 independent experiments. e Confocal microscopic images of TIGAR (green) and TAK1 (red) in PMs. Scale bars, 5 μm. Images were representative images from three independent experiments. f Western blot of TAK1 in BMDM lysates from WT and Tigar KO mice treated by LPS for the indicated times using the indicated antibodies, n = 3 samples. g RAW264.7 cells were transfected with Lenti-Con or Lenti-TIGAR for 72 h followed by LPS treatment with or without TAK1 inhibitor 5Z-7-OX (100 nM) for 30 min. Western blot analysis of cell lysates using the indicated antibodies. h Quantifications of the phosphorylation and total TAK1, IKK, and p65 in the indicated groups, n = 3 samples. i mRNA levels of pro-inflammatory genes in RAW264.7 cells followed by LPS treatment with or without TAK1 inhibitor 5Z-7-OX (100 nM) for 12 h ( n = 3). Data are expressed as mean ± SEM. d Two-tailed Student t -test. f Two-way ANOVA followed by the Bonferroni test. h , i One-way ANOVA followed by the Bonferroni test. All blot assays were repeated three times independently with similar results. Source data are provided as a Source Data file.

Article Snippet: Briefly, TAK1 kinase, ATP, and different doses of purified TIGAR protein (Ag17661, Protein Tech) were mixed in a kinase reaction buffer containing DTT for 30 min for completion of the kinase reaction.

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis

doi: 10.1038/s41467-024-48708-0

Figure Lengend Snippet: a BMDMs isolated from WT and Tigar KO mice were stimulated with LPS for 30 min and subjected to Co-IP with anti-TAK1 antibody followed by western blot with anti-Ub antibody. b HEK293 cells were transfected by His-TAK1, HA-Ub and Flag-TIGAR plasmids. Co-IP and western blot of the ubiquitination of TAK1. c HEK293 cells were transfected by His-TAK1, HA-Ub-K63O and Flag-TIGAR plasmids. Co-IP and western blot of K63 ubiquitination of TAK1. HA-Ub-K63O indicates ubiquitin in which all lysines except K63 were mutated. d BMDMs isolated from WT and Tigar KO mice were stimulated with LPS for 30 min and subjected to Co-IP with anti-TAK1 antibody followed by western blot with anti-K63-Ub antibody. e HEK293 cells were transfected by His-TAK1, HA-TRAF6 and Flag-TIGAR plasmids. Co-IP and western blot of the complex formation between TAK1 and TRAF6. f BMDMs isolated from WT and Tigar KO mice were stimulated with LPS for 30 min and subjected to Co-IP with anti-TRAF6 antibody followed by western blot with anti-TAK1 antibody. g Quantifications of the effect of TIGAR on the complex formation between TAK1 and TRAF6 ( n = 3). h HEK293 cells were transfected by indicated plasmids and TAK1 ubiquitination was detected by western blot. i , j Co-IP and western blot of the interactions between TIGAR, TAK1, and TRAF6 in HEK293 cells transfected by indicated plasmids. k HEK293 cells were transfected by Flag-TIGAR and HA-tagged TAK1 fragments. Co-IP and western blot of the binding between TIGAR and TAK1 fragments. l HEK293 cells were transfected by Flag-TIGAR and HA-tagged TRAF6 fragments. Co-IP and western blot of the binding between TIGAR and TRAF6 fragments. Data are expressed as mean ± SEM. g One-way ANOVA followed by the Bonferroni test. All blot assays were repeated three times independently with similar results. Source data are provided as a Source Data file.

Article Snippet: Briefly, TAK1 kinase, ATP, and different doses of purified TIGAR protein (Ag17661, Protein Tech) were mixed in a kinase reaction buffer containing DTT for 30 min for completion of the kinase reaction.

Techniques: Isolation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Binding Assay

Journal: Nature Communications

Article Title: Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis

doi: 10.1038/s41467-024-48708-0

Figure Lengend Snippet: a , b HEK293 cells were transfected by HA-TAK1, HA-TRAF6 and Flag-TIGAR fragments. Co-IP and western blot of the binding between TIGAR fragments and TAK1 ( a ) or TRAF6 ( b ). c Co-IP and western blot of the complex formation between TAK1 and TRAF6 after transfected by Flag-TIGAR fragments. d Co-IP and western blot of the ubiquitination of TAK1 after transfected by Flag-TIGAR fragments. e RAW264.7 cells were transfected by Lenti-Con, Lenti-TIGAR, and Lenti-TIGAR (1–210) for 72 h followed by LPS treatment for 12 h. mRNA levels of Tnf-a , Nos2 , Ccl2 , and Ccl8 in RAW264.7 cells, n = 3 samples. f Gibbs free energy landscape of the first two principal components (PCs) generated from MD simulations for the binding between TAK1 and TIGAR, where the dark blue color areas indicate lower energetic conformations. The lowest potential well highlighted with a gray circle. According to the TAK1 (pale green)-TIGAR (purple) binding mode extracted from the lowest potential well, the details of the critical molecular interaction between the two proteins have been zoomed in. g Wild type and three sets of designed mutant sequences of 146–161 residues of TIGAR for validating the predicted binding mode. h HEK293 cells were transfected by HA-TAK1 and Flag-tagged TIGAR mutants. Co-IP and western blot of the binding motif between TIGAR and TAK1. i HEK293 cells were transfected by HA-TAK1, His-TRAF6, and Flag-tagged TIGAR mutants. Co-IP and western blot of the complex formation between TAK1 and TRAF6 after transfected by Flag-tagged TIGAR mutants. j HEK293 cells were transfected by His-TAK1, HA-Ub and Flag-tagged TIGAR mutants. Co-IP and western blot of the ubiquitination of TAK1 after transfected by Flag-tagged TIGAR mutants. Data are expressed as mean ± SEM. e One-way ANOVA followed by the Bonferroni test. All blot assays were repeated three times independently with similar results. Source data are provided as a Source Data file.

Article Snippet: Briefly, TAK1 kinase, ATP, and different doses of purified TIGAR protein (Ag17661, Protein Tech) were mixed in a kinase reaction buffer containing DTT for 30 min for completion of the kinase reaction.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Ubiquitin Proteomics, Generated, Mutagenesis

Journal: Nature Communications

Article Title: Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis

doi: 10.1038/s41467-024-48708-0

Figure Lengend Snippet: Tigar KO mice were infected with myeloid-specific CD11b promoter-driven lentivirus (pCDH-CD11b-T2A-copGFP) encoding Flag-TIGAR, Flag-TMU, or Flag-Mut2, respectively. After 4 days the CLP sepsis model was generated. a Western blot of Flag-TIGAR, Flag-TMU and Flag-Mut2 expression in spleen F4/80 + cells. b Clinical score of mice was analyzed ( n = 7). c Murine survival rate was determined during 96 h of CLP challenge ( n = 12). d , e mRNA levels of pro-inflammatory genes in lung ( d ) ( n = 6), and spleen F4/80 + cells ( e ) ( n = 6). f Plasma concentrations of TNF-α, and CCL2 in mice ( n = 7). g Schematic diagram of the mechanism underlying 5Z-7-OX competing with TIGAR for binding to TAK1. Yellow structure: residues 152–157 of TIGAR; Blue structure: residues 158–161 of TIGAR. h HEK293 cells were transfected by HA-TAK1 and Flag-TIGAR plasmids. Co-IP and western blot of TIGAR-TAK1 complex formation in HEK293 cells incubated with 0.1, 0.3, or 1 mM 5Z-7-OX for 12 h. i HEK293 cells were transfected by HA-TAK1, His-TRAF6 and Flag-TIGAR plasmids and treated with or without 0.1 mM 5Z-7-OX. Co-IP and western blot of TAK1-TRAF6 complex formation in HEK293 cells. Male C57BL/6J mice were intraperitoneally injected with 5Z-7-OX or DMSO (Con). After 1 h, mice were induced with CLP sepsis and euthanized 12 h later. j Clinical score of mice was analyzed ( n = 8). k – m mRNA levels of pro-inflammatory genes in lung ( k ) ( n = 7), liver ( l ) ( n = 7), and spleen F4/80 + cells ( m ) ( n = 7). n Plasma concentrations of TNF-α, CCL2 and CCL8 in Con and 5Z-7-OX ( n = 7) treated mice. Data are expressed as mean ± SEM. b , d – f One-way ANOVA followed by the Bonferroni test. c Log-rank (Mantel–Cox) test. j Two-tailed Student t -test. k – n Two-tailed Student t -test, Two-tailed t -test with Welch correction, or two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.

Article Snippet: Briefly, TAK1 kinase, ATP, and different doses of purified TIGAR protein (Ag17661, Protein Tech) were mixed in a kinase reaction buffer containing DTT for 30 min for completion of the kinase reaction.

Techniques: Infection, Generated, Western Blot, Expressing, Clinical Proteomics, Binding Assay, Transfection, Co-Immunoprecipitation Assay, Incubation, Injection, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TIGAR regulates canonical NF-κB signaling. NT, TKD, and TOE 3T3-L1 preadipocytes were generated as described under “Method details.” A, 3T3-L1 adipocytes were either left untreated (Basal) or stimulated with 10 ng/ml TNFα for 5 min (TNFα). Cell lysates were prepared and immunoblotted for the indicated proteins. These are representative immunoblots independently performed five times. B and C, the adipocytes were either left untreated or stimulated with 10 ng/ml TNFα for 4 h, and the expression of Ccl2 (B) and A20 (C) mRNAs was determined by qRT-PCR. These data represent the average of five independent determinations ± S.D. (error bars). D, the TKD 3T3-L1 cells were stably infected with TWT and TMU lentiviruses as described under “Method details.” The 3T3-L1 TKD, TWT, and TMU preadipocytes were differentiated into adipocytes either left untreated (Basal) or stimulated with 10 ng/ml TNFα for 5 min (TNFα). Cell lysates were prepared and immunoblotted for the indicated proteins. These are representative immunoblots independently performed three times. E and F, the TKD, TWT, and TMU adipocytes were treated with vehicle or 10 ng/ml TNFα for 4 h, and the expression of Ccl2 (E) and A20 (F) mRNAs was determined by qRT-PCR. These data represent the average of three independent determinations ± S.D. *, p < 0.05; ****, p < 0.0001.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Generated, Western Blot, Expressing, Quantitative RT-PCR, Stable Transfection, Infection

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TWT and TMU inhibit NF-κB pro-inflammatory gene expression, IKKβ phosphorylation, and IKKβ enzymatic activity. A, HEK293T cells were seeded in a 6-well plate (0.8 × 106 cells/well) in 2 ml of growth medium (10% fetal bovine serum with antibiotics) for 6–8 h. Following transfection with the NF-κB luciferase reporter gene, relative basal and TNFα-stimulated levels of luciferase activity were determined as described under “Method details.” These data are the average of three independent determinations ± S.D. (error bars). B and C, HEK293T cells were transfected with cDNA for IKKβ alone or in combination with TWT or TMU cDNAs, as described under “Method details.” Ccl2 (B) and A20 (C) mRNA levels were determined by qRT-PCR, and these data represent the average of three independent determinations ± S.D. D, the HEK293T cells were transfected with the indicated combinations of cDNAs (20 μg total/100-mm dish) for 24 h, followed by immunoblotting of cell lysates for the indicated proteins as described under “Method details.” Lane 1, 20 μg of pcDNA; lane 2, 10 μg of TWT; lane 3, 10 μg of TMU; lane 4, 5 μg of IKKβ; lane 5, 10 μg of IKKβ plus 10 μg of TWT; lane 6, 10 μg of IKKβ plus 10 μg of TMU cDNA plus various amounts of empty vector for a total of 20 μg of DNA. The extracts were then immunoblotted for the various proteins indicated. These are representative immunoblots independently performed three times. E, HEK293T cells (6-well plate, 0.8 × 106 cells/well) were transfected with 2.5 μg of empty vector (Ctrl), 1 μg of IKKβ, 1.5 μg of TWT, 1.5 μg of TMU, 2.5 μg of IKKβ plus TWT, and IKKβ plus TMU cDNAs for 24 h. Cell extracts were prepared, and IKKβ kinase activity in vitro was determined using IκBα as substrate as described under “Method details.” F, cell extracts were prepared, and equal amounts of IKKβ and TWT cell extracts were premixed (IKKβ + TWT) for 0.5 h before determination of IKKβ kinase activity. The data are the average ± S.E. from four independent experiments, each performed in duplicate.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Gene Expression, Phospho-proteomics, Activity Assay, Transfection, Luciferase, Quantitative RT-PCR, Western Blot, Plasmid Preparation, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TIGAR inhibits IKKβ-dependent phosphorylation of several direct IKKβ cellular substrate targets. A, HEK293T cells were transfected with the indicated combinations of cDNAs for 24 h followed by immunoblotting of cell lysates for the indicated proteins as described under “Method details.” Lane 1, 4 μg of empty vector; lane 2, 2 μg of TIGAR; lane 3, 1 μg of IKKβ; lane 4, 1 μg of NEMO; lane 5, 2 μg of TIGAR plus 1 μg of IKKβ; lane 6, 1 μg of IKKβ plus 1 μg of NEMO; lane 7, 2 μg of TIGAR plus 1 μg of NEMO; lane 8, 2 μg of TIGAR plus 1 μg of IKKβ plus 1 μg of NEMO cDNA plus various amounts of pcDNA for a total of 4 μg of DNA/60-mm dish for 24 h. These are representative immunoblots independently performed 3–5 times. B, HEK293T cells were transfected with 3 μg of empty vector (lane 1), 1 μg of IKKβ (lane 2), 1 μg of IKKβ (lane 3) plus increasing amounts of TIGAR cDNA (0.25 μg (lane 3), 0.5 μg (lane 4), 1 μg (lane 5), and 2 μg (lane 6)) plus various amounts of empty vector for a total of 3 μg/60-mm dish of DNA. 24 h later, cell extracts were prepared and immunoblotted for the indicated proteins. These are representative immunoblots independently performed 3–5 times. C, the TIGAR dose-dependent inhibition of IKKβ phosphorylation from the data obtained in B was quantified by ImageJ densitometry ± S.D. (error bars) as described under “Method details.” D, the TIGAR dose-dependent inhibition of CYLD phosphorylation (band indicated by arrow) from the data obtained in Fig. 3B was quantified by ImageJ densitometry ± S.D. (error bars) as described under “Method details.”

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Phospho-proteomics, Transfection, Western Blot, Plasmid Preparation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: NEMO rescues the TIGAR inhibition of IKKβ-dependent signaling. A, HEK293T cells were transfected with 4 μg of empty vector (lane 1), 1 μg of IKKβ (lane 2), 1 μg of IKKβ plus 2 μg of TIGAR (lane 3) with increasing amounts (0.125 μg (lane 4), 0.25 μg (lane 5), 0.5 μg (lane 6), and 1 μg (lane 7)) of NEMO plus various amounts of empty vector for a total of 4 μg/60-mm dish of DNA. 24 h later, cell extracts were prepared and immunoblotted for the indicated proteins. This is a representative immunoblot independently performed 3–5 times. B, the NEMO dose-dependent rescue of TWT inhibition of IKKβ phosphorylation from the data obtained in A was quantified by ImageJ densitometry ± S.D. (error bars). C, the NEMO dose-dependent rescue of TWT inhibition of CYLD phosphorylation from the data obtained in A was quantified by ImageJ densitometry ± S.D. D, the NEMO dose-dependent rescue of TWT inhibition of Ccl2 gene expression from the cells transfected with cDNA was performed as in Fig. 4A. Ccl2 mRNA was determined by qRT-PCR from two independent experiments. The data are presented as the average ± S.D. ns, not significant; ##, p < 0.01; ###, p < 0.001 comparing IKKβ versus IKKβ + TIGAR; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 comparing IKKβ + TIGAR versus increasing amounts of NEMO.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Inhibition, Transfection, Plasmid Preparation, Western Blot, Phospho-proteomics, Gene Expression, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TIGAR specifically co-immunoprecipitates with HOIP, the E3 ligase subunit of LUBAC. A, HEK293T cells were transfected with 3 μg of TIGAR or 3 μg of TIGAR plus 3 μg of FLAG-HOIP, 3 μg of TIGAR plus 3 μg of FLAG-HOIL-1L, or 3 μg of TIGAR plus 3 μg of FLAG-SHARPIN cDNAs on 100-mm plates for 18 h. Aliquots of the cell lysates were immunoblotted with the FLAG and TIGAR antibodies. B, the cell lysates in A were immunoprecipitated (IP) with the FLAG antibody and immunoblotted for FLAG and TIGAR. These are representative immunoblots independently performed two times.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TIGAR and NEMO compete for HOIP binding. A, HEK293T cells were transfected with the indicated combinations of cDNAs (8 μg total/100-mm dish) for 16 h, followed by immunoblotting of cell lysates for the indicated proteins. Lane 1, 8 μg of empty vector; lane 2, 3 μg of HOIP; lane 3, 2 μg of TIGAR; lane 4, 1 μg of the NEMO; lane 5, 3 μg of HOIP plus 2 μg of TIGAR; lane 6, 3 μg of HOIP plus 1 μg of NEMO; lanes 7–9, 3 μg of HOIP plus 2 μg of TIGAR with increasing amounts of NEMO (0.1, 0.3, 1, and 3 μg, respectively). B, the cell lysates from A were immunoprecipitated (IP) with a FLAG antibody and immunoblotted for the indicated proteins. These are representative immunoblots independently performed two times.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Binding Assay, Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: Identification of the HOIP amino acid sequences responsible for TIGAR binding. A, schematic representation of the HOIP linear amino acid sequence with known protein interaction domains and various deletion mutants generated. The values to the right of each model structure reflect the percentage of HOIP mutant binding to TIGAR compared with full-length HOIP determined as described below. The percentage of TIGAR binding was normalized for the relative levels of HOIP expression. B, HEK293T cells were transfected with TIGAR and HA-NEMO with full-length and NZF1 domain–deleted FLAG-HOIP cDNAs, as indicated. The cell lysates were immunoblotted for FLAG-HOIP, HA-NEMO, and TIGAR (left, lanes 1–9). The cell lysates were immunoprecipitated (IP) with the FLAG antibody, and these immunoprecipitates were immunoblotted for FLAG-HOIP, HA-NEMO, and TIGAR (right, lanes 1–9). These are representative immunoblots independently performed 2–3 times. C, HEK293T cells were transfected with TIGAR and the ZF, NZF1, NZF2, and UBA domain-deleted FLAG-HOIP cDNAs, as indicated. The cell lysates were immunoblotted for FLAG-HOIP and TIGAR (left, lanes 1–6). The cell lysates were immunoprecipitated with the FLAG antibody, and these immunoprecipitates were immunoblotted for FLAG-HOIP and TIGAR (right, lanes 1–6). These are representative immunoblots independently performed three times. D, cells were transfected with TIGAR and various N-terminal deletion FLAG-HOIP cDNAs, as indicated. The cell lysates were immunoblotted for FLAG-HOIP and TIGAR (left, lanes 1–5). The cell lysates were immunoprecipitated with the FLAG antibody, and these immunoprecipitates were immunoblotted for FLAG-HOIP and TIGAR (right, lanes 1–5). These are representative immunoblots independently performed three times. E, cells were transfected with TIGAR and various C-terminal deletion FLAG-HOIP cDNAs, as indicated. The cell lysates were immunoblotted for FLAG-HOIP and TIGAR (left, lanes 1–6). The cell lysates were immunoprecipitated with the FLAG antibody, and these immunoprecipitates were immunoblotted for FLAG-HOIP and TIGAR (right, lanes 1–6). These are representative immunoblots independently performed three times.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Binding Assay, Sequencing, Generated, Mutagenesis, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TIGAR suppresses LUBAC-induced NEMO linear ubiquitination. A, HEK293T cells were transfected with HOIP-FLAG, HOIL-1L-FLAG, SHARPIN-FLAG, UBE2L3-FLAG (2.5 μg each), and either TWT or TMU (10 μg each) plus various amounts of empty vector for a total of 20 μg of cDNA/100-mm dish. 24 h later, cell lysates were prepared and immunoblotted for the indicated proteins and total M1 linear ubiquitinated proteins, as described under “Method details.” Lane 1, pcDNA; lane 2, TWT; lane 3, TMU; lane 4, LUBAC plus UBE2L3; lane 5, LUBAC plus UBE2L3 and TWT; lane 6, LUBAC plus UBE2L3 and TMU cDNAs. These are representative immunoblots independently performed three times. B, the lysates (500 μg) from A were immunoprecipitated (IP) with a NEMO-specific antibody (5 μg) and immunoblotted with the linear ubiquitination-specific (M1 Ub), NEMO, and HOIP antibodies. These are representative immunoblots independently performed three times. C, 3T3-L1 NT, TKD, and TOE adipocytes were either left untreated (Basal) or stimulated with 10 ng/ml TNFα for 5 min. Cell lysates were prepared and immunoblotted for the indicated proteins. The cell lysates (800 μg) were also immunoprecipitated with NEMO-specific antibody, and the NEMO immunoprecipitates were immunoblotted with the linear ubiquitination-specific (M1 Ub) and NEMO antibody. These are representative immunoblots independently performed three times.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: NEMO overexpression rescues the TIGAR inhibition of linear ubiquitination. A, HEK293T cells were transfected with the various indicated cDNAs composed of HOIP, SHARPIN, HOIL-1L, UBEL3 cDNAs (LUBAC), TWT, TMU, and NEMO cDNAs (4 μg each) plus various amounts of empty vector for at a total of 20 μg/100-mm dish for 24 h. Cell lysates were immunoblotted for the indicated proteins as described under “Methods details.” B, the cell lysates in A were immunoblotted with the M1 linear ubiquitination (M1 Ub) antibody. These are representative immunoblots independently performed two times.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: Over Expression, Inhibition, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC

doi: 10.1074/jbc.RA118.002727

Figure Lengend Snippet: TIGAR regulates adipose tissue NF-κB signaling in vivo. C57Bl6/J male WT, whole-body TKO, and TGRS mice at 12 weeks of age were maintained on a low-fat diet as described under “Experimental procedures.” A, two independent control WT and TKO male mice were given an intraperitoneal injection of vehicle or TNFα (10 μg/kg) for 15 min. The epididymal adipose tissue was extracted and immunoblotted for TIGAR, total IκB, pSer32/36-IκB, pSer536-RelA, total RelA, pSer418-CYLD, total CYLD, and actin as loading control. These are representative immunoblots independently performed three times. WT, wildtype mice from TKO inbreds. B, two independent control WT and TGRS mice were given an intraperitoneal injection of vehicle or TNFα for 15 min. The epididymal adipose tissue was extracted and immunoblotted for TIGAR, total IκB, pSer32/36-IκB, pSer536-RelA, total RelA, pSer418-CYLD, total CYLD, and actin as loading control. These are representative immunoblots independently performed two times. C, epididymal adipose tissue from control WT, TKO, and TGRS mice treated with and without TNFα for 15 min were subjected to qRT-PCR for Ccl2 mRNA expression. These data are the average of four independent determinations ± S.D. (error bars). ****, p < 0.0001, TKO-TNF versus WT-TNF and TGRS-TNF versus WT-TNF.

Article Snippet: The full-length human TIGAR cDNA (SC320794, Origene) was used as a template to produce DNA fragment with the addition of BamHI and XbaI sites, which were then cloned into a pCR-Blunt II-TOPO vector (Life Technologies, Inc.).

Techniques: In Vivo, Control, Injection, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cell systems

Article Title: Four key steps control glycolytic flux in mammalian cells

doi: 10.1016/j.cels.2018.06.003

Figure Lengend Snippet:

Article Snippet: TIGAR: TIGAR (D3F4A) Rabbit mAb , Cell Signaling , Cat# 14751.

Techniques: Recombinant, Modification, Transfection, Cloning, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Lactate Dehydrogenase Assay, Bicinchoninic Acid Protein Assay, PK Assay, Plasmid Preparation, Sequencing, Expressing, Variant Assay, Cell Culture

Journal: Developmental Cell

Article Title: TIGAR Is Required for Efficient Intestinal Regeneration and Tumorigenesis

doi: 10.1016/j.devcel.2013.05.001

Figure Lengend Snippet: Increased TIGAR Expression in Primary Human Colon Cancer and Associated Metastases (A) Expression of TIGAR in various human cancer cell lines with different p53 status. (B) Example of TIGAR staining in matched samples of human normal colon, colon adenocarcinoma, and liver metastasis from the same patient. (C) Quantification of the staining intensity in normal human colon, primary adenocarcinoma (tumor), and liver metastases.

Article Snippet: TMA of human cancer patients was obtained from AccuMax Array (A203(II)-colon cancer tissues liver metastasis) and was stained with anti-human TIGAR antibody (ProSci).

Techniques: Expressing, Staining

Journal: International journal of molecular medicine

Article Title: miR‑199a‑3p/Sp1/LDHA axis controls aerobic glycolysis in testicular tumor cells.

doi: 10.3892/ijmm.2018.3771

Figure Lengend Snippet: Figure 5. Sp1 Si588 and miR‑199a‑3p suppress the LDHA expression. (A) The mRNA expression and (B) protein expression of 4 metabolic genes (LDHA, PGK1, MCT1 and TIGAR) were detected in Ntera‑2 cells following treatment with miR‑199a‑3p mimics or Sp1 Si588, respectively. *P≤0.05 and **P≤0.01 vs. NC group. miRNA, microRNA; MiNC, miRNA negative control mimics; SiNC, siRNA negative control; Sp1, specificity protein 1; siRNA, small inter- fering RNA; LDHA, lactate dehydrogenase A; TIGAR, tumor protein 53 induced glycolysis regulatory phosphatase; PGK1, phosphoglycerate kinase 1; MCT1, solute carrier family 16 member 1.

Article Snippet: The following primary antibodies were incubated at 4 ̊C for 12 h: Sp1 (1:500; cat. no., CSB‐PA050124; Cusabio Technology LLC, Wuhan, China), LDHA (1:2,000; cat. no. 19987‐1‐AP; Wuhan Sanying Biotechnology, Wuhan, China), TIGAR (1:1,000, cat. no., CSB‐PA162542, Cusabio Technology LLC), MCT1 (1:1,000; cat. no., sc‐365501; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), PGK1 (1:1,000; cat. no., CSB‐PA00035A0Rb; Cusabio Technology LLC), actin (1:10,000; cat. no., AC026; ABclonal, Woburn, MA, USA) and GAPDH (1:5,000; cat. no., AP0063; Bioworld Technology, Inc., St. Louis, MN, USA).

Techniques: Expressing, Negative Control