Journal: PLoS ONE

Article Title: A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

doi: 10.1371/journal.pone.0001372

Figure Lengend Snippet: A. TRADD, C/EBPβ, RIP and caspase 8 immunoblots were performed on C/EBPβ immunoprecipitates from primary human HSC treated with an ERK1/2 inhibitor (10 µM) or the cell permeant Ac-KA217VD-CHO peptide (200 µM). Blocking the phosphorylation of C/EBPβ by RSK with the ERK1/2 inhibitor or the cell permeant Ac-KA217VD-CHO (KAVD) peptide, increased the association between C/EBPβ, active caspase 8, TRADD and RIP. β-Actin was used as an internal control for the immunoprecipitations. B. TNFR1 and C/EBPβ immunoblots were performed on C/EBPβ immunoprecipitates from HSC isolated from mice treated with CCl 4 for 12 or 16 weeks as described in . Blocking the phosphorylation of C/EBPβ by RSK with C/EBPβ-Ala217 transgene or the cell permeant Ac-KA217VD-CHO peptide increased the association between C/EBPβ and TNFR1. β-Actin was used as an internal control for the immunoprecipitations. C. TRAF2 and C/EBPβ immunoblots were performed on C/EBPβ immunoprecipitates from HSC isolated from mice treated with CCl 4 for 12 or 16 weeks as described in . Blocking the phosphorylation of C/EBPβ by RSK with C/EBPβ-Ala217 transgene or the cell permeant Ac-KA217VD-CHO peptide increased the association between C/EBPβ and TRAF2. β-Actin was used as an internal control for the immunoprecipitations. D. Cytochrome C and Apaf1 immunoblots were performed on cytochrome C immunoprecipitates in livers from mice treated with CCl 4 for 12 or 16 weeks as described in . Blocking the phosphorylation of C/EBPβ by RSK with C/EBPβ-Ala217 transgene or the cell permeant Ac-KA217VD-CHO peptide increased the association between cytochrome C and Apaf1. β-Actin was used as an internal control for the immunoprecipitations.

Article Snippet: Pre-cleared stellate cell lysates were incubated for 2 h with purified C/EBPβ, RSK, TRADD or caspase 8 antibodies followed by the addition of A/G+ agarose (Santa Cruz Biotechnology) for 12 h. The immunoprecipitation reactions each contained 500 μg of total protein and 2 μg antibody (or purified IgG pre-immune serum as negative control).

Techniques: Western Blot, Blocking Assay, Phospho-proteomics, Control, Isolation

Journal: Antioxidants

Article Title: Anti-Amnesic Effect of Walnut via the Regulation of BBB Function and Neuro-Inflammation in Aβ 1-42 -Induced Mice

doi: 10.3390/antiox9100976

Figure Lengend Snippet: List of antibodies and their information used in this study.

Article Snippet: TNFR1 , CSB-PA621879EA01HU , 1:1000 , Cusabio (Hubei, China).

Techniques:

Journal: Antioxidants

Article Title: Anti-Amnesic Effect of Walnut via the Regulation of BBB Function and Neuro-Inflammation in Aβ 1-42 -Induced Mice

doi: 10.3390/antiox9100976

Figure Lengend Snippet: Protective effect of walnut ( Juglans regia L.) extract on Aβ-induced neuro-inflammation: ( A ) protein expression levels; ( B ) representative Western blots for total protein and expression of tumor necrosis factor-alpha (TNF-α) ( B ), tumor necrosis factor receptor 1 (TNFR1) ( C ), phosphorylated c-Jun N-terminal kinase (p-JNK) ( D ), phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (p-IκB) ( E ), cyclooxygenase-2 (COX-2) ( F ), and interleukin 1 beta (IL-1β) ( G ) in mice brain tissues. Results shown are means ± SD ( n = 3). Data are statistically represented at * = significantly different from the NC group; # = significantly different from Ab group, respectively; * and # p < 0.05.

Article Snippet: TNFR1 , CSB-PA621879EA01HU , 1:1000 , Cusabio (Hubei, China).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation

doi: 10.3390/ijms222212459

Figure Lengend Snippet: RIPK1 prevented TNF-dependent TRADD modification and degradation independent of cIAP and could be prevented by A20 over-expression. Control cells and RIPK1 KO clones were treated ( A ) with TNF for the indicated time points. ( B ) The cells from ( A ) were pretreated with BTZ and CHX for 5 h before stimulation with TNF for the indicated time points. ( C ) The cells from ( A ) were pretreated with IAP antagonist before stimulation with TNF for 2 h. ( D ) Control and RIPK1 KO cells were transduced with control or A20 containing LV and stimulated with TNF for the indicated time points. Protein expression was analyzed by WB. The WB shown are representative of at least two independent experiments.

Article Snippet: The following antibodies (Abs) were used for WB: RIPK1 (R41220), TRADD (T50320), and FADD (F36620) Abs were purchased from BD Transduction Laboratories, San Diego, California.

Techniques: Modification, Over Expression, Clone Assay, Transduction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation

doi: 10.3390/ijms222212459

Figure Lengend Snippet: Suggested model of TRADD and RIPK1 functions in TNF signaling and ripoptosome formation. Under normal conditions (yellow field), the signal initiated by TNF through TNF-R1 proceeds via the assembly of TNF complex I and initiates NF-κB and MAPK signaling. Assembly of complex I and stabilization of NIK are the requirements for ripoptosome formation, which can direct the cell to apoptosis or necroptosis. NIK stabilization can be blocked by cIAPs or TRADD. When RIPK1 is missing (green field) complex I is assembled but NF-κB and MAPK signaling are partially blocked. No ripoptosome is formed. The assembly of pseudocomplex is unable to direct the cell-to-cell death. Unknown E3 ubiquitin ligase can ubiquitinate TRADD and direct it to proteasomal degradation. When TRADD is missing (red field), complex I consists only of unmodified RIPK1 and both NF-κB and MAPK signaling are partially blocked. NIK stabilization is simplified and ripoptosome is assembled and can direct the cell to apoptosis or necroptosis.

Article Snippet: The following antibodies (Abs) were used for WB: RIPK1 (R41220), TRADD (T50320), and FADD (F36620) Abs were purchased from BD Transduction Laboratories, San Diego, California.

Techniques:

Journal: Scientific Reports

Article Title: Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

doi: 10.1038/s41598-017-03211-z

Figure Lengend Snippet: Deficiency of TRADD induces impaired DNA damage response. ( a ) Western blotting analysis shows blotting for γH2AX, TRADD, and Actin in TRADD +/+ and TRADD −/− MEF cells treated with H 2 O 2 in time-dependent manner (0.5 mM). ( b ) γH2AX foci (Red) were analyzed in TRADD +/+ and TRADD −/− MEF cells treated with H 2 O 2 (0.5 mM) by Immunofluorescense as described in A. Scale bars, 10 μm. ( c ) Immunofluorescence analyses of γH2AX (Green) in H 2 O 2 (0.5 mM) treated TRADD +/+ and TRADD −/− MEF for 2 hours (upper panels) and release from H 2 O 2 treated TRADD +/+ and TRADD −/− MEF for 4 hours (lower panels). Cells were stained with anti-γH2AX (Green) and DAPI (Blue). Scale bars, 10 μm. ( d ) Western blotting analysis shows results consistent with immunofluorescence as described in ( c ). ( e ) After cells were treated with etoposide (25 μM) for 1 hour, TRADD +/+ and TRADD −/− MEF replaced with fresh media. Cells were stained with anti-γH2AX (Red) and DAPI (Blue). Western blotting analysis (lower panel) shows the consistent results with immunofluorescence. Scale bars, 10 μm. ( f ) Quantitative analysis of γH2AX foci was conducted in TRADD knock-downed U2OS cells. After TRADD knockdown, cells were treated with phleomycin (Phleo) and then stained with γH2AX antibody. *P < 0.05 (Student’ s t-test). ( g ) Transient knockdown of TRADD induces unrepaired DNA damage in HeLa cells. Western blot analysis shows γH2AX status in response to H 2 O 2 in TRADD KD HeLa cells. Cells were transfected with siRNA TRADD or siRNA negative control (NC), respectively. After 48 hours, the cells were continuously treated with H 2 O 2 (0.5 mM). The whole cell lysates were analysed by western blot as using indicated antibodies. ( h ) Reconstitution of TRADD in TRADD −/− MEFs. Western blotting analysis shows γH2AX expression in response to continuous treatment with H 2 O 2 (0.5 mM) in different time points.

Article Snippet: TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex.

Techniques: Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, Negative Control, Expressing

Journal: Scientific Reports

Article Title: Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

doi: 10.1038/s41598-017-03211-z

Figure Lengend Snippet: DNA damage induces nuclear translocation of TRADD. ( a ) HeLa cells were transiently transfected with GFP-TRADD and treated with H 2 O 2 (0.5 mM) for indicated time points. Cells were analyzed by confocal fluorescence microscopy. ( b ) HeLa cells were transiently transfected with GFP-TRADD and treated with H 2 O 2 (0.5 mM). After treatment, live cell Images were analyzed by confocal fluorescence microscopy for 70 minutes (left panel). Quantitative analysis of nuclear translocation of TRADD was measured by GFP intensity in the nucleus (right panel). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (Student’s t-test). ( c ) Colocalization of GFP-TRADD and mCherry-FokI at single DNA double-strand break site. GFP empty vector (EV), GFP-TRADD wild type (WT), or GFP-TRADD Src mutant (SRC) was cotranfected with mCherry-FokI (mCh-FokI) nuclease into U2OS 2-6-3 cell lines. After 48 hr, cells were fixed and stained with DAPI for nuclear staining. Images were analyzed confocal microscope (Nikon A1). Scale bar, 10 μm. ( d ) HeLa cell were transiently transfected with NES mutant TRADD and treated with H 2 O 2 (0.5 mM) or MNNG (0.25 mM) for indicated times. Cells were fractionated into cytoplasmic and nuclear fractions using an NE-PER fractionation kit. Anti-Hsp90 or anti-Sp1 used as a control for normalization of cytoplasm and nuclear lysates, respectively. ( e ) Western blotting analysis was conducted with lysates from TRADD −/− (MOCK), NES-mutant TRADD (NES-TRADD) and Src-myristoylation-TRADD (Src-TRADD) in TRADD −/− MEFs treated with H 2 O 2 (0.5 mM) for indicated time periods (left panel). Expression of γH2AX was analyzed in TRADD −/− and TRADD −/− (NES-mutant TRADD) MEFs treated with H 2 O 2 (0.5 mM) for 1 hour using immunofluorescence (right panels).

Article Snippet: TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex.

Techniques: Translocation Assay, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Mutagenesis, Staining, Fractionation, Control, Western Blot, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

doi: 10.1038/s41598-017-03211-z

Figure Lengend Snippet: TRADD is required for non-homologous end-joining repair. ( a ) Knockdown efficacy for TRADD in DNA repair reporter cell lines EJ5 and DR. ( b ) After 48 hours transfection with TRADD, RPA80, or BRCA1 targeting siRNAs into reporter cell lines, each siRNA was again cotransfected with an I- SceI endonuclease construct. After 72 hours, GFP positive cells were analyzed with a flow cytometer (FACScan). **P < 0.01; ***P < 0.001; n.s., not significant (ANOVA). ( c , d ) After 1 hour with laser microirradiation, endogenous NHEJ repair factors were stained with each antibody at DNA break sites: 53BP1 ( c ); Ku70/80 and 53BP1 ( d ). Scale bars, 10 μm. ( e , f ) Endogenous HR repair factors were stained as described in c . RAD51 ( e ); RPA32 and RAD51 ( f ). γH2AX was used as a DNA damage marker at DNA break sites in ( c ) and ( e ). Scale bars, 10 μm. ( g ) The protein levels of repair factors in TRADD depletion. EJ-5 or DR cells were transfected with TRADD siRNAs (#1 or #2) or control siRNA. The levels of repair factors were detected by using target antibody, respectively. Total protein levels were verified with Ponceus S staining and tubulin antibody as a loading control.

Article Snippet: TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex.

Techniques: Non-Homologous End Joining, Knockdown, Transfection, Construct, Flow Cytometry, Staining, Marker, Control