Journal: Oncotarget

Article Title: Interactome analysis of transforming growth factor-β-activated kinase 1 in Helicobacter pylori -infected cells revealed novel regulators tripartite motif 28 and CDC37

doi: 10.18632/oncotarget.24544

Figure Lengend Snippet: Immunoblots of TAK1 ( A ), TAB2 ( B ), TAB3 ( C ) immunoprecipitates, subcellular fractions ( D ) and cellular lysates ( E ) are represented. The cells were exposed to H. pylori , TNF or IL-1β for the indicated times (in min). H. pylori lysate (Hp) was used to demonstrate the absence of antibody cross-reactivity with bacterial proteins. In some lysates of the 30 min-infected cells, the immunoprecipitation antibody was not added to approve non-specific protein binding to the beads. The arrows point specific protein bands. (E) Transfection with TAK1 siRNA was performed 48 h prior to stimulation. C, cytosol, CL, cell lysate; Cskn, cytoskeleton; IP, immunoprecipitation; M, membranes; N, nuclei.

Article Snippet: TAB2 knock-out cells were selected from AGS cells transfected with TAB2 CRISPR/Cas9 KO and HDR plasmids (2 μg of each, Santa Cruz Biotechnology Inc.) using puromycin.

Techniques: Western Blot, Infection, Immunoprecipitation, Protein Binding, Transfection

Journal: Oncotarget

Article Title: Interactome analysis of transforming growth factor-β-activated kinase 1 in Helicobacter pylori -infected cells revealed novel regulators tripartite motif 28 and CDC37

doi: 10.18632/oncotarget.24544

Figure Lengend Snippet: AGS cells were transfected with siRNAs against TAK1 ( A ) and TAB1 ( B , D ) or with TAB2 CRISPR/Cas9 and HDR plasmids ( C , E ). The cells were exposed to H. pylori , TNF or IL-1β for the indicated times (in min). (A–C) Cell lysates were prepared and analyzed via immunoblotting. Densitometry analysis of represented immunoblots was performed. The band intensities (in %) in TAB1 knockdown and TAB2 knockout cells relative to the respective mock values within the infection time course are indicated. (D, E) Cells were transfected with luciferase reporters, treated with H. pylori or cytokines and collected with Passive Lysis buffer for the Transactivation assay. ** p < 0.01 vs non-stimulated cells, # p < 0.05 and ## p < 0.01 vs stimulated mock cells.

Article Snippet: TAB2 knock-out cells were selected from AGS cells transfected with TAB2 CRISPR/Cas9 KO and HDR plasmids (2 μg of each, Santa Cruz Biotechnology Inc.) using puromycin.

Techniques: Transfection, CRISPR, Western Blot, Knockdown, Knock-Out, Infection, Luciferase, Lysis, Transactivation Assay

Journal: Oncotarget

Article Title: Interactome analysis of transforming growth factor-β-activated kinase 1 in Helicobacter pylori -infected cells revealed novel regulators tripartite motif 28 and CDC37

doi: 10.18632/oncotarget.24544

Figure Lengend Snippet: ( A ) AGS cells were transfected with TAK1 or (and) TAB1 DNA plasmids for 24 h, cellular lysates were prepared from non-starved cells and analyzed by immunoblotting. ( B ) Endogenous TAK1 was immunoprecipitated from mock- or TAB1-transfected AGS cells. ( C ) TAK1 was overexpressed and immunoprecipitated from untreated, H. pylori -infected (for 20 and 45 min) and TAB1-co-expessing cells. The samples (B, C) were analyzed by MS. The size of the prey nodes reflects the number of bait interactions, i.e., the smallest nodes have one interaction and the largest interact with all baits. Prey that interact with all baits were positioned at the center of the network; the gray nodes represent novel interactors of interest. TAB1, TAB2 and TAB3 nodes are highlighted in yellow. The already known TAK1 interactors, which have been experimentally validated in human cells and are annotated in the databases InnateDB and IntactDB, are highlighted in red. ( D ) Schematic domain structure of TAK1, TAB1 and TAB2 with phosphorylated (blue), glycosylated (orange) and acetylated (green) sites detected by MS. Interactions between TAK1, TAB1 and TAB2 are shown with dotted lines. BR, binding region; CUE, coupling of ubiquitin conjugation to endoplasmic reticulum; ZF RanBP2, zinc finger domain found in Ran binding protein 2.

Article Snippet: TAB2 knock-out cells were selected from AGS cells transfected with TAB2 CRISPR/Cas9 KO and HDR plasmids (2 μg of each, Santa Cruz Biotechnology Inc.) using puromycin.

Techniques: Transfection, Western Blot, Immunoprecipitation, Infection, Binding Assay, Ubiquitin Proteomics, Conjugation Assay

Journal: Oncogene

Article Title: Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells.

doi: 10.1038/onc.2011.627

Figure Lengend Snippet: Figure 1 (a) Immunoblotting analysis of Tab2 in TamR cells as compared with MCF7 wt responding to IL-1b treatment. TamR cells were transfected with control or Tab2 siRNA and kept in 1% DC-FBS for 48 h. MCF7 wt cells were kept in 1% DC-FBS for 3 days then treated with 10 ng/ml IL-1b for 10 min. MCF7 wt and TamR cells were subjected to cellular fractionation and the Tab2 protein analyzed by immunoblotting in the cytoplasmatic and nuclear fractions. (b) Tab2 downregulation restores tamoxifen response in TamR cells. MCF7 wt and TamR cells were transfected with control or Tab2 siRNA and 48 h later treated with 108 M E2 (white bars) or 108 M E2 plus 106 M 4OHT (black bars). After 24 h, the proliferation was measured by 2 h bromo-deoxyuridine incorporation. Data are means±s.d. of replicate experiments (MCF7wt and TamR-4.2 N ¼ 4; TamR-4.1 N ¼ 3; TamR-8 N ¼ 5). *Denotes Pp0.05 (Mann–Whitney U test). More details are given as Supplementary Figure S2.

Article Snippet: We used also a polyclonal anti-Tab2 antibody from Bethyl Laboratories (Montgomery, TX, USA) and a monoclonal anti-Hsp90 from Stressgene Bioreagents Corp. (Victoria, BC, Canada).

Techniques: Western Blot, Transfection, Control, Cell Fractionation, MANN-WHITNEY

Journal: Oncogene

Article Title: Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells.

doi: 10.1038/onc.2011.627

Figure Lengend Snippet: Figure 2 In vivo and in vitro interaction between Tab2 and ERa. (a) Coimmunoprecipitation of Tab2 and Flag-tagged ERa. Full length Tab2 and Flag-tagged ERa were transiently overexpressed in HEK293T cells. Anti-Flag and anti-Tab2 immunoprecipitates were carried out from nuclear extracts and were analyzed by immunoblotting with anti-Flag and anti-Tab2 antibodies. (b) Competition of ERa peptide for Tab2/ERa interaction. MBP pull-down assays, using full length Tab2 or the Tab2 central domain (aa 406-531) fused to MBP, and ERa overexpressed in HEK293T cells, in the presence of ERa-peptide or ERa mut-peptide. (c) Amino-acid sequences of ERa-TAT peptide and mutated ERa-TAT peptide. The vertical bars indicate amino-acid changes.

Article Snippet: We used also a polyclonal anti-Tab2 antibody from Bethyl Laboratories (Montgomery, TX, USA) and a monoclonal anti-Hsp90 from Stressgene Bioreagents Corp. (Victoria, BC, Canada).

Techniques: In Vivo, In Vitro, Western Blot

Journal: Oncogene

Article Title: Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells.

doi: 10.1038/onc.2011.627

Figure Lengend Snippet: Figure 3 An ERa mimic peptide restores tamoxifen response in TamR cells. (a) TamR cells were treated with 1 mM or 100 mM ERa- TAT peptide (black bars) or ERa mut-TAT peptide (white bars) in absence of serum. After 1 h, 1% DC-FBS and 106 M 4OHT were added for 24 h, then cell growth was measured as described in Figure 1. Data are means±s.d. of triplicate experiments. (b) ERa- TAT peptide competition on Tab2/ERa interaction in vivo. Full length Tab2 and Flag-tagged ERa were transiently overexpressed in HEK293T cells. The cells were treated with 100 mM of ERa-TAT or ERa mut-TAT peptides for 3 or 6 h in the presence of 4OHT. Anti-Flag immunoprecipitates from nuclear extracts were analyzed by immunoblotting with anti-Flag and anti-Tab2 antibodies. ‘m’ indicates untransfected HEK293T lysate. The ratio between anti- Tab2 and anti-Flag for each treatment is indicated. (c) BT474 cells were treated with ERa-TAT peptide (black bars) or ERa mut-TAT peptide (white bars) and their growth was analyzed as above. Data are from triplicate experiments. (d) BT474 cells were transfected with control or Tab2 siRNA and then treated with 108 M E2 (white bars) or 108 M E2 plus 106 M 4OHT (black bars). Data are means±s.d. of duplicate experiments. *Denotes Pp0.05 as in Figure 1.

Article Snippet: We used also a polyclonal anti-Tab2 antibody from Bethyl Laboratories (Montgomery, TX, USA) and a monoclonal anti-Hsp90 from Stressgene Bioreagents Corp. (Victoria, BC, Canada).

Techniques: In Vivo, Western Blot, Transfection, Control

Journal: Oncogene

Article Title: Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells.

doi: 10.1038/onc.2011.627

Figure Lengend Snippet: Figure 4 Top network of Tab2 siRNA-regulated genes (IPA, Ingenuity Systems). Down (green) and up (red) regulated genes are connected with continuous or dashed lines, indicating direct interactions experimentally proven and indirect connections, respectively. Basc indicates the BRCA1 associated complex.

Article Snippet: We used also a polyclonal anti-Tab2 antibody from Bethyl Laboratories (Montgomery, TX, USA) and a monoclonal anti-Hsp90 from Stressgene Bioreagents Corp. (Victoria, BC, Canada).

Techniques:

Journal: Allergy, Asthma & Immunology Research

Article Title: Overexpression of miR-155-5p Inhibits the Proliferation and Migration of IL-13-Induced Human Bronchial Smooth Muscle Cells by Suppressing TGF-β-Activated Kinase 1/MAP3K7-Binding Protein 2

doi: 10.4168/aair.2018.10.3.260

Figure Lengend Snippet: TAB2 is a direct target of miR-155-5p. (A) Search in the online bioinformatics tool Targetscan indicated that 3′-UTR of TAB2 mRNA is a potential target of miR-155-5p. (B) qRT-PCR experiments indicated that IL-13 dose-dependently induced the expression of TAB2 mRNA in BSMCs. (C) Western blot analysis indicated that IL-13 dose-dependently induced the expression of TAB2 protein in BSMCs. (D) Luciferase reporter assay indicated TAB2 is a direct target of miR-155-5p. (E) miR-155-5p mimic significantly inhibited the expression of TAB2. TAB2, transforming growth factor (TGF)-β-activated kinase 1/MAP3K7-binding protein 2; UTR, untranslated region; qRT-PCR, quantitative real-time polymerase chain reaction; IL, interleukin; BSMC, bronchial smooth muscle cell; NC, negative control; ns, not significant. * P <0.05, † P <0.01, ‡ P <0.001, and § P <0.0001 when compared with control or NC.

Article Snippet: The human transforming growth factor (TGF)-β-activated kinase 1/MAP3K7-binding protein 2 (TAB2) small interfering RNA (siRNA) (h) was purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Control

Journal: Allergy, Asthma & Immunology Research

Article Title: Overexpression of miR-155-5p Inhibits the Proliferation and Migration of IL-13-Induced Human Bronchial Smooth Muscle Cells by Suppressing TGF-β-Activated Kinase 1/MAP3K7-Binding Protein 2

doi: 10.4168/aair.2018.10.3.260

Figure Lengend Snippet: Silencing of TAB2 mimics the effects of miR-155-5p on BSMCs. (A) MTT assay was performed to evaluate the effects of TAB2 siRNA and miR-155-5p mimic on the proliferation of IL-13-induced BSMCs. Transfection with miR-155-5p mimic significantly inhibited cell proliferation. The same effect was found with TAB2 siRNA. (B) Transwell migration assay was performed to evaluate the effects of TAB2 siRNA and miR-155-5p mimic on the proliferation of IL-13-induced BSMCs. Transfection with miR-155-5p mimic significantly inhibited cell migration. The same effect was found with TAB2 siRNA. TAB2, transforming growth factor (TGF)-β-activated kinase 1/MAP3K7-binding protein 2; BSMC, bronchial smooth muscle cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; IL, interleukin; ns, not significant. * P <0.05, † P <0.01, ‡ P <0.001, § P <0.0001; ∥ P <0.05 and ¶ P <0.001 when compared with the control siRNA+IL-13 group.

Article Snippet: The human transforming growth factor (TGF)-β-activated kinase 1/MAP3K7-binding protein 2 (TAB2) small interfering RNA (siRNA) (h) was purchased from Santa Cruz Biotechnology.

Techniques: MTT Assay, Transfection, Transwell Migration Assay, Migration, Binding Assay, Small Interfering RNA, Control