Journal: Oncogene

Article Title: Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals.

doi: 10.1038/sj.onc.1209576

Figure Lengend Snippet: Figure 1 GSTP1-1 inhibits TNF-a-induced ASK1–JNK pathway activation. HeLa cells were transiently transfected with hemagglu- tinin (HA)-GSTP1-1 (0.3 and 1.0 mg) or empty expression vectors. After 30 h, the transfected cells were treated with ( þ ) or without () TNF-a (50 ng/ml) for 30 min. Cell lysates were subjected to immunoblot (IB) analysis with anti-phospho- JNK (Thr183/Tyr185), anti-phospho-MKK4 (Ser257/Thr261), anti-phospho-ASK1 (Thr845), anti-IkB, anti-GSTP1-1 and control antibodies as indicated. Phosphorylation of kinases was deter- mined. Relative kinases phosphorylation fold is shown as mean of duplicates from three independent experiments, with untreated as 1.0.

Article Snippet: Mouse monoclonal antibody against HA-tag and polyclonal antibodies against JNK/SAPK, MKK4, phospho-JNK/SAPK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-MKK4 (Ser257/Thr261), phospho-MKK7 (Ser271/ Thr275) and phospho-ASK1 (Thr845) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Transfection, Expressing, Western Blot, Control, Phospho-proteomics

Journal: Oncogene

Article Title: Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals.

doi: 10.1038/sj.onc.1209576

Figure Lengend Snippet: Figure 2 GSTP1-1 physically interacts with TRAF2 in vivo and in vitro. (a) Lysates from HeLa cells treated with TNF-a (50 ng/ml) were subjected to immunoprecipitation (IP) using indicated antibodies (anti-JNK1, anti-p38a, anti-MKK4, anti-MKK6, anti-ASK1, anti-MEK kinase 1, anti-TRAF2 and anti-TRAF6, 0.5 mg each) or control normal immunoglobulin G (IgG). The precipitates were analysed by immunoblotting with GSTP1-1 monoclonal antibody and corresponding antibodies. The total amounts of GSTP1-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the whole-cell lysates were determined by immunoblot analysis (bottom panel). (b) HEK293 cells were cotransfected with hemagglutinin (HA)-tagged GSTP1-1 (1 mg) and Flag-tagged TRAF2 (0.5 mg) or empty expression vectors as indicated. After 36 h, the cell lysates (500 mg) were immunoprecipitated with EzviewTM red anti-Flag M2 affinity gel (5 ml), followed by immunoblot analysis with anti-HA antibody or anti-TRAF2 antibody, and the whole-cell lysates were also immunoanalysed with anti-HA antibody. (c) HEK293 cells were co-transfected with HA-GSTP1-1 (0.5 mg, lanes 2 and 3), Flag- TRAF2 (1 mg, lane 2) and Flag-TRAF6 (1 mg, lane 3) as indicated. The whole-cell lysates were subjected to immunoprecipitation using anti-HA antibody and analysed by immunoblotting with anti-Flag antibody to verify the coprecipitated Flag-TRAF2 or Flag-TRAF6. The presence of overexpressed exogenous proteins in the same lysates was verified by immunoblotting. (d) Lysates (1 mg) of HEK293 cells transfected with Flag-tagged TRAF2 (upper panel) or TRAF6 (lower panel) were incubated with His-GSTP1-1 immobilized on Ni2 þ–IDA–agarose beads in the lysis buffer at 41C for 8 h. Immunoblotting of the bound proteins with anti-Flag antibody was shown. One representative experiment is shown in each panel.

Article Snippet: Mouse monoclonal antibody against HA-tag and polyclonal antibodies against JNK/SAPK, MKK4, phospho-JNK/SAPK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-MKK4 (Ser257/Thr261), phospho-MKK7 (Ser271/ Thr275) and phospho-ASK1 (Thr845) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, In Vitro, Immunoprecipitation, Control, Western Blot, Expressing, Transfection, Incubation, Lysis

Journal: Oncogene

Article Title: Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals.

doi: 10.1038/sj.onc.1209576

Figure Lengend Snippet: Figure 6 GSTP1-1 inhibits TRAF2-induced ASK1 catalytic activity. (a) HEK293 cells were co-transfected with the indicated combinations of expression vectors producing HA-ASK1 (0.1 mg), Flag-TRAF2 (2 mg) and Xpress-GSTP1-1 (1 mg). The cell lysates (1 mg) were immunoprecipitated with anti-HA antibody (0.5 mg), and the immunopellets were detected by immunoblot analysis with anti-phospho-ASK1 (Thr845) antibody. (b) HEK293 cells were transiently co-transfected with HA-tagged ASK1 (1 mg), Flag- tagged TRAF2 (1.5 mg) and Xpress-tagged GSTP1-1 (0.5, 1.0 and 1.5 mg) as indicated. After 36 h, the HA-tagged ASK1 were immunoprecipitated with anti-HA antibody (0.5 mg) from 200 mg of whole-cell lysates, and their kinase activities (KA) were assessed using His-MKK7 (K149M) as substrate. Equal amount of HA- tagged ASK1 used in each assay were confirmed by immunoblot- ting. Data shown are the representative of three independent experiments. The fold activation relative to that of cells co- transfected with an empty vector and an expression vector encoding HA-tagged ASK1 is the average of the three experiments. (c) HEK293 cells were co-transfected with Xpress-tagged GSTP1-1 (1 mg), Flag-tagged TRAF2 (1.5 mg) and HA-tagged ASK1 (1 mg) as indicated. After 36 h, the cells lysates (600 mg) were immunopreci- pitated with EzviewTM red anti-Flag M2 affinity gel or anti-Xpress antibody (0.5 mg), and the immunopellets were analysed by immunoblot analysis with anti-HA antibodies. Overexpressed ASK1 and GSTP1-1 were detected using anti-HA and anti-Xpress antibodies, respectively. One representative experiment is shown in each panel.

Article Snippet: Mouse monoclonal antibody against HA-tag and polyclonal antibodies against JNK/SAPK, MKK4, phospho-JNK/SAPK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-MKK4 (Ser257/Thr261), phospho-MKK7 (Ser271/ Thr275) and phospho-ASK1 (Thr845) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Activation Assay, Plasmid Preparation

Journal: Oncogene

Article Title: Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals.

doi: 10.1038/sj.onc.1209576

Figure Lengend Snippet: Figure 7 Effects of GSTP1-1 on TNF-a-induced interaction between TRAF2 and ASK1. (a) Characterization of hGSTP1-1 small interfering RNA (siRNA). HeLa cells were transfected with either RNAiFectTM transfection reagent alone (left lane), 100 nM non- silencing siRNA (middle lane) or hGSTP1-1-specific siRNA (right lane) as described in Materials and methods. Cell lysates (20 mg) were subjected to immunoblotting using monoclonal anti-GSTP1 antibody to determine the efficiency of GSTP1-1 suppression (upper panel) and GAPDH expression (bottom panel). (b) HeLa cells were transfected with either control siRNA (lanes 1–12) or GSTP1-1 siRNA (lanes 13–18). After 36 h, the cells were transfected again with the same amount of siRNA together with HA tagged GSTP1-1 expression vector (lanes 7–12) or empty vector (lanes 1–6 and 13–18). At 36 h after the second transfection, the cells were treated with TNF-a (50 ng/ml) for the indicated periods. Cell lysates were subjected to immunoblot analysis with anti-phospho-JNK (Thr183/ Tyr185) and anti-phospho-ASK1 (Thr845) antibodies. The same lysates were immunoprecipitated with anti-TRAF2 antibody followed by immunoblot analysed with anti-ASK1 or anti-MEK kinase 1 antibodies. The efficiency of GSTP1-1 overexpression (lanes 7–12) and suppression (lanes 13–18) was determined using anti-GSTP1-1 antibody. (c) The transfected HeLa cells were treated with TNF-a (50 ng/ml) and cycloheximide (CHX, 1 mg/ml) for 12 h. Phase-contrast photography of transfected HeLa cells treated with or without TNF-a/CHX as indicated. (d) The transfected HeLa cells were incubated with 5 mM N-acetyl-L-cysteine (NAC) for 5 h followed by treated with TNF-a/CHX for 12 h. Apoptotic cell death was assessed by flow cytometry as described in Material and methods. The percentage of apoptotic cells are shown as means7s.d. from three independent experiments.

Article Snippet: Mouse monoclonal antibody against HA-tag and polyclonal antibodies against JNK/SAPK, MKK4, phospho-JNK/SAPK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-MKK4 (Ser257/Thr261), phospho-MKK7 (Ser271/ Thr275) and phospho-ASK1 (Thr845) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Small Interfering RNA, Transfection, Western Blot, Expressing, Control, Plasmid Preparation, Immunoprecipitation, Over Expression, Incubation, Cytometry

Journal: Oncogene

Article Title: Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals.

doi: 10.1038/sj.onc.1209576

Figure Lengend Snippet: Figure 8 Binding affinities of GSTP1-1 mutants to TRAF2. (a) Diagrams of the putative TRAF2-binding motifs (TWQE) in wild- type hGSTP1-1. (b) HEK293 cells were co-transfected with the indicated expression vectors, and after 36 h, the cells lysates were immunoprecipitated with anti-Xpress antibody, followed by immunoblot analysis with anti-Flag antibody. (c) Lysates of HEK293 cells (1 mg protein) expressing Flag-tagged TRAF2 were incubated with wild-type His-GSTP1-1 and its mutants immobilized on Ni2 þ– IDA–agarose beads in the lysis buffer at 41C for 8 h, respectively. The bound proteins were washed and eluted followed by immunoblot analysis using anti-Flag antibody. His-GSTP1-1 proteins were stained by Coomassie blue. (d) HeLa cells were co-transfected with 5 Jun2-Luc (0.2 mg), Flag-tagged TRAF2, Xpress-tagged GSTP1-1 wild-type or mutants as indicated. The luciferase activities were measured and values were normalized based on b-galactosidase (b-gal) activities. Values shown are averages (mean7s.d.) of one representative experiment in which each transfection was performed in triplicate. (e) HEK293 cells were co-transfected with indicated expression vectors, and after 36 h, the cells were lysed. The HA-tagged ASK1 was immunoprecipitated with anti-HA antibody (0.5 mg) from 200 mg of lysates, and the kinase activities (KA) were assessed using His-MKK7 (K149M) as substrate. (f) HEK293 cells were co- transfected with indicated expression vectors. After 36 h, cell apoptosis were assessed by flow cytometry. The graph shows the percentage of apoptotic cells. Data shown are averages of three independent experiments.

Article Snippet: Mouse monoclonal antibody against HA-tag and polyclonal antibodies against JNK/SAPK, MKK4, phospho-JNK/SAPK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-MKK4 (Ser257/Thr261), phospho-MKK7 (Ser271/ Thr275) and phospho-ASK1 (Thr845) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Incubation, Lysis, Staining, Luciferase, Cytometry

Journal: Cells

Article Title: Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation

doi: 10.3390/cells10113171

Figure Lengend Snippet: A2AR and A1AR pharmacological stimulation of primary steatotic mouse HP: effects on HR injury, OS production, TRAF2 levels and ASK1/JNK activation. Control (HP) or steatotic hepatocytes (S-HP) pre-exposed or not to the A2AR agonist CGS21680 (5 µM), to the A1R agonist CCPA (100 µM) or to the antioxidant DPPD (5 μM) were stored at 4 °C in hypoxic conditions for 8 h in presence (S-HP) or absence (HP) of palmitic acid (PA, 50 μM) and then reoxygenated at 37 °C for 30 min. ( A ) Cell viability, ( B ) Visualization of lipid intracellular content, ( C ) Oxidant species (OS) production, ( D ) TRAF2 expression, ( E ) ASK1 activation, ( F ) JNK activation. The different parameters were estimated after normoxic incubation (O2), and/or at the end of hypoxic storage (To) and after 15 and/or 30 min of reoxygenation (HR). The results are expressed as mean of 3–5 experiments ± SD. Statistical significance: * § # p < 0.05, ## p < 0.01.

Article Snippet: ASK1 activation, which was evaluated in liver sections that were stained with the ASK1 (phospho-Thr845) antibody (Biorbyt, Cambridge, UK) (dilution 1:200), was considered positive when the staining of the HPs was detectable in the cytoplasm, independently from the intensity of the staining.

Techniques: Activation Assay, Control, Expressing, Incubation

Journal: Cells

Article Title: Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation

doi: 10.3390/cells10113171

Figure Lengend Snippet: Effects of A2AR and A1AR pharmacological stimulation on HR injury and ASK1/JNK activation of steatotic C1C7 cells with downregulated expression of ASK1. Steatotic (PA 700 μM, S-C1C7) and non steatotic C1C7 cells (C1C7) transfected with control SiRNA or ASK1 SiRNA and pre-exposed or not to the A2AR agonist CGS21680 (5 µM) or to the A1R agonist CCPA (100 µM), were stored at 4 °C for 8 h in hypoxic conditions in in presence (S-C1C7) or absence (C1C7) of palmitic acid (PA, 700 μM) and reoxygenated at 37 °C for 30 min (HR30′). ( A ) WB analysis of ASK1 expression after C1C7 cells transfection with Control SiRNA or ASK1 SiRNA (SiRNA); ( B ) Cell viability; ( C ) JNK activation. The different parameters were estimated after normoxic incubation (O2), and/or at the end of hypoxic storage (T0), or after 30 min of reoxygenation (HR). The results represent the mean of 3 experiments ± SD. Statistical significance: * # § p < 0.05.

Article Snippet: ASK1 activation, which was evaluated in liver sections that were stained with the ASK1 (phospho-Thr845) antibody (Biorbyt, Cambridge, UK) (dilution 1:200), was considered positive when the staining of the HPs was detectable in the cytoplasm, independently from the intensity of the staining.

Techniques: Activation Assay, Expressing, Transfection, Control, Incubation

Journal: Cells

Article Title: Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation

doi: 10.3390/cells10113171

Figure Lengend Snippet: Inhibition of OS production or of PI3K/Akt activation in steatotic HP treated with A2AR or A1R agonist and exposed to H/R: effect on viability, on activation of Akt, ASK1 and JNK and on inhibition of ASK1. Control (HP) and steatotic hepatocytes (S-HP) pre-exposed or not to the A2AR agonist CGS21680 (5 µM) or to the A1R agonist CCPA (100 µM), in presence or absence of the antioxidant DPPD (5 μM) or of the PI3K/Akt inhibitor wortmannin (WM, 5 nM), were stored at 4 °C for 8 h in hypoxic VIASPAN solution added (S-HP) or not (HP) with palmitic acid (PA, 50 μM) and then reoxygenated at 37 °C for 30 min. ( A ) Akt activation. ( B ) ASK1 inhibition, ( C ) Cell viability, ( D ) ASK1 activation, ( E ) JNK activation. The different parameters were estimated after 15 or 30 min of reoxygenation (H/R). The results are expressed as mean of 3–5 experiments ± SD. Statistical significance: * # § p < 0.05.

Article Snippet: ASK1 activation, which was evaluated in liver sections that were stained with the ASK1 (phospho-Thr845) antibody (Biorbyt, Cambridge, UK) (dilution 1:200), was considered positive when the staining of the HPs was detectable in the cytoplasm, independently from the intensity of the staining.

Techniques: Inhibition, Activation Assay, Control

Journal: Cells

Article Title: Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation

doi: 10.3390/cells10113171

Figure Lengend Snippet: Effects of pharmacological A2AR and A1R stimulation on hepatic IRI of steatotic mice. Mice fed with normal (ND) or high fat diet (HFD) for 9 weeks, treated or not by I.P. injection with the A2aR agonist, CGS21680 (0.5 mg/kg) or with the A1R agonist CCPA (1.5 mg/kg), were subjected to 45 min of ischemia followed by 120 min of reperfusion (IR). ( A ) Hepatic triglyceride content ( B ) ALT release ( C ) Akt activation ( D ) Visualization of activated ASK1 ( E ) ASK1 inhibition; ( F ) ASK1 activation; ( G ) JNK activation. The results represent the mean of 3 experiments ± SD. Statistical significance: * # § p < 0.05.

Article Snippet: ASK1 activation, which was evaluated in liver sections that were stained with the ASK1 (phospho-Thr845) antibody (Biorbyt, Cambridge, UK) (dilution 1:200), was considered positive when the staining of the HPs was detectable in the cytoplasm, independently from the intensity of the staining.

Techniques: Injection, Activation Assay, Inhibition