Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 2Cβ Association with the IκB Kinase Complex Is Involved in Regulating NF-κB Activity

doi: 10.1074/jbc.m306273200

Figure Lengend Snippet: FIG. 1. PP2C associates with IKK complex. A, Western blot analysis with the FLAG antibody was performed on extracts prepared from G418-resistant 293 cells either in the absence (lane 1) or presence of FLAG-PP2C (lane 2). B, extracts from the 293 cells stably express- ing FLAG-tagged PP2C were immunoprecipitated (IP) with either normal mouse IgG (lane 1) or the FLAG antibody (lane 2) followed by Western blot analysis with mouse monoclonal antibodies directed against IKK (top) and IKK (middle) or a rabbit polyclonal antibody for IKK/NEMO (bottom). The extract input is also shown (lane 3). C, a CMV expression vector alone (lane 1) or encoding Myc-tagged wild type PP2C (2.0 g) was cotransfected into 293 cells with 2.0 g of either FLAG-tagged IKK (lane 2), IKK (lane 3), or IKK/NEMO (lane 4). Extracts were immunoprecipitated with either the FLAG antibody (top) or normal mouse IgG (middle), and Western blot analysis was per- formed with a Myc antibody to detect Myc-PP2C (lanes 1–5, top and middle). Western blot analysis of these extracts with the FLAG and Myc antibodies was also performed (lanes 1–4, bottom). D, extracts from HeLa cells were immunoprecipitated with either normal mouse IgG (lane 1) or a murine polyclonal antibody directed against PP2C (lane 2) followed by Western blot analysis with antibodies directed against IKK (top), IKK (middle), and IKK/NEMO (bottom). The extract input is also shown (lane 3). The bottom shows Western blot analysis of extracts prepared from HeLa cells either in the absence (lane 1) or presence of a transfected FLAG-PP2C construct (lane 2) using the murine polyclonal antibody directed against PP2C.

Article Snippet: Antibodies used in these studies included monoclonal antibodies directed against the M2 FLAG epitope (F-3165; Sigma), the Myc epitope (sc-40; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), IKK (556532; Pharmingen), and IKK (550621; Pharmingen) in addition to normal mouse IgG (sc-2025; Santa Cruz Biotechnology) and polyclonal antibodies directed against I B (sc-371; Santa Cruz Biotechnology), phosphoI B (9241; Cell Signaling), IKK (sc-7607; Santa Cruz Biotechnology), IKK /NEMO (sc-8330; Santa Cruz Biotechnology), IKK (sc-7218; Santa Cruz Biotechnology), influenza hemagglutinin (HA) epitope (sc805; Santa Cruz Biotechnology), or actin (A2066; Sigma).

Techniques: Western Blot, Stable Transfection, Immunoprecipitation, Bioprocessing, Expressing, Plasmid Preparation, Transfection, Construct

Journal: Frontiers in Immunology

Article Title: Fbxo16 mediates degradation of NF-κB p65 subunit and inhibits inflammatory response in dendritic cells

doi: 10.3389/fimmu.2025.1524110

Figure Lengend Snippet: Fbxo16 forms a CRL1 complex, binds to p65 and negatively regulates NF-κB signaling. (A) Interaction of Fbxo16 and Cullins in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-Flag, and immunoblotted with anti-Cullin 1, Cullin 2 or Cullin 3. (B) Interaction of Fbxo16 and Skp1 in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Skp1. (C) Interaction of Fbxo16 and p65 in whole cell lysates of HEK293T cells, transfected with a Flag-p65 or p50 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-p65 or p50. (D) Interaction of Fbxo16 and PDLIM2 in whole cell lysates of HEK293T cells, transfected with a Flag-PDLIM2 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Flag. (E) Luciferase assay in MEFs transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs along with increasing amounts (wedge) of Fbxo16, then left untreated or stimulated with LPS for 5 hr. (F) Luciferase assay in HEK293T cells transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs without or with MyD88, IKKβ, TRAF6 or p65 along with increasing amounts (wedge) of Fbxo16. Data are representative of three (A, B) , four (C, D) or three ( E, F ; means ± SD, **P<0.01) independent experiments.

Article Snippet: Expression plasmids for Flag-tagged murine p50 (#20018), murine TRAF6 (#21624), murine MyD88 (#13093) and human IKKβ (#23298) were purchased from Addgene.

Techniques: Transfection, Immunoprecipitation, Luciferase, Construct

Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) HEK293 cells were transiently transfected with a His-3xFLAG-FAT10 (FLAG-FAT10) expression construct and stimulated for 24 h with TNF. Lysates were subjected to immunoprecipitation using FLAG-reactive antibodies, coupled to sepharose beads, and subsequently analyzed by Phos-tag/SDS–PAGE/IB analysis. Where indicated, cells were pretreated before TNF stimulation with the displayed inhibitors for a total of 3 h (10 µM each). (B) HEK293 cells were transiently transfected with expression plasmids for the different kinases. Cells were harvested, lysed, and subjected to immunoprecipitation using anti-FLAG or anti-HA antibodies, coupled to sepharose beads. Subsequently, the immunoprecipitated kinases were incubated with recombinant FAT10 (rFAT10) and an in vitro reaction was performed in the kinase buffer. The phosphorylation status of FAT10 was analyzed by Phos-tag/SDS–PAGE and IB. Asterisks mark unspecific background bands. (C) FLAG-FAT10 and the indicated kinases were transiently overexpressed in HEK293 cells followed by TNF stimulation. After 24 h, cells were lysed and subjected to immunoprecipitation against the FLAG-tag, combined with Phos-tag/SDS–PAGE and IB analysis. (D) Recombinant FAT10 (rFAT10) was incubated with recombinant kinases IKKβ, IKKε or MK3 for 45 min at 30°C. Subsequently, proteins were separated on a Phos-tag/SDS–PAGE followed by immunoblot analysis using the antibodies indicated. (E) HEK293 cells were prepared as described in (A). Where specified, cells were pretreated with the inhibitors indicated (10 µM each) for a total of 3 h before stimulation with TNF. One representative example out of three independent experiments with the same outcomes is shown. Source data are available for this figure.

Article Snippet: When indicated, cells were pretreated for 3 h with the following inhibitors: 10 μM IKK-16 (pan-IKK inhibitor, S2882; Selleckchem), 10 μM SP600125 (pan-JNK inhibitor, S1460; Selleckchem), 10 μM SB203580 (p38 MAPK inhibitor, S1076; Selleckchem), 10 μM trametinib (MEK1/2 inhibitor, S2673; Selleckchem), 5 μM TPCA-1 (IKKβ inhibitor, SC-203083; Santa Cruz) or 10 μM CAY10576 (IKKε inhibitor, 10011249; Cayman).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Incubation, Recombinant, In Vitro, Phospho-proteomics, FLAG-tag, Western Blot

Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) FAT10 mRNA levels were analyzed and quantified by real-time PCR in A549 cells 24 h after TNF/IFNγ treatment or 24 h after IAV infection. (B) A549 cells were infected with IAV (MOI:1) for 0, 6, 24 or 48 h, as indicated. Cell lysates were subjected to SDS–PAGE combined with immunoblot analysis with the antibodies indicated. (C) Immunoblot showing endogenous FAT10 expression in A549 cells treated for 24 h with TNF/IFNγ, or infected for 24 h with IAV. FAT10 was immunoprecipitated with a monoclonal FAT10-reactive antibody (clone 4F1) and an immunoblot was performed with the antibodies indicated. (D) A549 cells stably expressing His-3xFLAG-FAT10 (FLAG-FAT10) were lysed after 24 h of TNF stimulation or after 24 h of IAV infection. The lysates were subjected to immunoprecipitation using FLAG-reactive antibodies coupled to sepharose beads. Subsequently, a Phos-tag/SDS–PAGE and immunoblot analysis with the indicated antibodies was performed. Asterisk marks an unspecific background band. (E) Recombinant 6His-SUMO-FAT10 was purified via Ni-NTA and left bound to the beads. 400 units λ phosphatase was added and incubated twice for 30 min at 30°C. Beads were extensively washed and FAT10 was eluted by treatment with the ULP1 enzyme to receive untagged and non-phosphorylated FAT10. Lysates from untreated or IAV-infected A549 cells were prepared and incubated with the purified FAT10, as indicated, for 30 min at 30°C. Proteins were separated on a Phos-tag/SDS–PAGE and analyzed by immunoblotting with a FAT10-reactive, polyclonal antibody. (F) A549 cells stably expressing FLAG-FAT10 were infected with IAV for 24 h. Cell lysates were subjected to a FLAG-immunoprecipitation and analyzed by Phos-tag/SDS–PAGE, followed by immunoblotting using the antibodies indicated. Where indicated, cells were pretreated with the indicated inhibitors (5 mM IKKβ inhibitor, 10 μM IKKε inhibitor) for 3 h, before IAV infection. For each panel, one representative example out of three independent experiments with similar outcomes is shown. Source data are available for this figure.

Article Snippet: When indicated, cells were pretreated for 3 h with the following inhibitors: 10 μM IKK-16 (pan-IKK inhibitor, S2882; Selleckchem), 10 μM SP600125 (pan-JNK inhibitor, S1460; Selleckchem), 10 μM SB203580 (p38 MAPK inhibitor, S1076; Selleckchem), 10 μM trametinib (MEK1/2 inhibitor, S2673; Selleckchem), 5 μM TPCA-1 (IKKβ inhibitor, SC-203083; Santa Cruz) or 10 μM CAY10576 (IKKε inhibitor, 10011249; Cayman).

Techniques: Real-time Polymerase Chain Reaction, Infection, SDS Page, Western Blot, Expressing, Immunoprecipitation, Stable Transfection, Recombinant, Purification, Incubation

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: A . A silver-stained polyacrylamide gel resolving the affinity-purified IKK complex. B . MS/MS spectra of [M+2H] 2+ ions of the peptides derived from the protein band corresponding to Hsp60. C . Immunoblot (IB) analyses of IKK subunits and Hsp60 in the affinity-purified complex. D . Interaction of Hsp60 with IKK complex. IKK complex was immunoprecipitated (IP) from HeLa cell lysates (500 µg total proteins for each) with IKKα, IKKβ, and IKKγ-specific antibodies. The IKKα/β/γ subunits, Hsp60, and Hsp90 were immunoblotted. WCL, whole cell lysate. E . TNF-α-independent interaction of Hsp60 and IKK complex. F . Co-immunoprecipitation of Hsp60 and IKK complex in cytosolic fraction. Upper panel , post-nuclear supernatant (PNS), cytosol (Cyto), and mitochondria (Mito) fractions from HeLa cells were immunoblotted. COX4 and tubulin were used as mitochondrial and cytosolic markers, respectively. Lower panel , Hsp60 was immunoprecipitated from the cytosolic fraction using either control goat IgG or anti-Hsp60 antibodies (K-19 and N-20). Representative blots are shown ( n = 3).

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Staining, Affinity Purification, Tandem Mass Spectroscopy, Derivative Assay, Western Blot, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: HeLa cells were immunoreacted with no primary ( A ), anti-Hsp60 ( B ), anti-IKKα ( C ), anti-IKKβ ( D ), anti-Hsp60/IKKα ( E ), and anti-Hsp60/IKKβ ( F ) antibodies, and then labeled with the corresponding secondary antibodies conjugated with 20 nm or 40 nm gold particles, as described in Experimental Procedure. The labeling was assessed by immuno-gold electron microscopy. Nuclei (Nu) and mitochondria (M) are indicated. Arrows indicate direct adherence of Hsp60- and IKK-labeled gold particles. No immunoreactive signal was seen in the sample without primary antibodies ( A ). The experiments were repeated twice with the same results, and representative results are shown.

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Labeling, Electron Microscopy

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: A . Direct binding of Hsp60 and IKK subunits. The 293T cells were co-transfected with Hsp60c (HA tag) and each of IKK subunit proteins (Flag tag) for 24 hrs. B . In vitro association of Hsp60 with IKKα and IKKβ. GST-fused Hsp60 proteins bound to the glutathione Sepharose beads was incubated with the lysates of Sf9 insect cells expressing His 6 -tagged IKK proteins. Hsp60 and IKKs were detected by immunoblotting for GST and HA tags, respectively. C . Schematic diagram showing deletion mutants of Hsp60. The putative phosphorylation sites of kinases, including PKA/PKG (1) and PKC (2), are indicated. D and E . Interaction of Hsp60 wild-type (WT) and deletion mutants with ectopically-expressed IKKα in 293T cells ( D ) or to endogenous IKK complex in HeLa cells ( E ). The control vector (C) is indicated. Representative blots and images are shown ( n = 3). n.s., nonspecific.

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Binding Assay, Transfection, FLAG-tag, In Vitro, Incubation, Expressing, Western Blot, Phospho-proteomics, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: A . Ablation of cytosolic Hsp60 by antisense ODNs. The cytosolic and mitochondrial fractions prepared from mock or ODN-transfected HeLa cells were immunoblotted. S, sense ODN; AS-1 and AS-2, antisense ODNs. The mitochondrial fraction was loaded at a volume of one-fifth of the corresponding cytosolic fraction. In particular, Prx III, which is an antioxidant enzyme present in the mitochondrial matrix, was used as mitochondrial markers to watch the nonspecific mitochondrial rupture. B . Half-life of ectopically-expressed Hsp60c protein (HA tag) after inhibition of protein synthesis with cycloheximide. The intensity of HA band was measured and normalized by the amount of IKKα band. Data in the graph are means ± S.D. of two independent experiments and fitted in SigmaPlot 8.0 software. C . Proteasome-dependent turnover of cytosolic Hsp60c protein. HeLa cells were pretreated with or without MG132 (5 µM) 30 min before cycloheximide treatment. D . TNF-α-induced IKK and JNK1 activation in mock or ODN-transfected cells. The in vitro kinase activity (KA) was averaged with the values from two independent experiments, and it is represented as a fold increase of the activity versus the unstimulated and mock-transfected cells (lane 1). E . NF-κB transcriptional activation in mock or ODN-transfected cells. The increasing concentration of AS-ODN (100 nM or 200 nM) was tested. The relative luciferase activity was normalized to the β-galactosidase activity and data are means ± S.D. of four independent experiments (* P <0.0001, ** P <0.001 versus stimulated S-ODN-transfected cells).

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Transfection, Inhibition, Software, Activation Assay, In Vitro, Activity Assay, Concentration Assay, Luciferase

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: A . Transduction of Hsp60-neutralizing antibody (Hsp60N) into the cytoplasm of HeLa cells. Mitotracker Red (Molecular Probes, USA) and DAPI indicate mitochondria and nuclei, respectively. B . The transduced Hsp60N antibody bound to endogenous Hsp60. After antibody transfection, the HeLa cell lysates were subjected to precipitation using Protein-A Sepharose beads. The precipitated proteins were immunoblotted for Hsp60. C . IKK and JNK1 activation in response to TNF-α in control IgG or Hsp60N antibody-transfected HeLa cells. The in vitro kinase activity (KA) was averaged with the values from two independent experiments, and it is represented as a fold increase of the activity versus the unstimulated and control IgG-transfected cells (lane 1). D . TNF-α-induced NF-κB transcriptional activation in antibody-transfected cells (* P <0.01 versus stimulated IgG-transfected cells).

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Transduction, Transfection, Activation Assay, Control, In Vitro, Activity Assay

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: Cells were transfected with either control (CGN) or Hsp60c-encoding plasmid (HA tag) for 24 hrs and then treated with TNF-α. A . Incorporation of ectopically-expressed Hsp60c (HA tag) in IKK complex. B . TNF-α-induced IKK activation in HeLa cells. C TNF-α-induced NF-κB activation in HeLa cells ( n = 4, * P <0.0001 versus unstimulated counterpart). D . TNF-α-induced NF-κB activation in the transfected IKKβ −/− 3T3 cells ( n = 4, * P <0.001 versus stimulated CGN-transfected cells, N.D. not detected).

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Transfection, Control, Plasmid Preparation, Activation Assay

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: A . Association of Hsp60c wild-type (WT) and mutants with IKKα and IKKβ. The indicated proteins were co-expressed in 293T cells as shown in . B and C . IKK ( B ) and NF-κB transcriptional activation ( C ) in cells expressing Hsp60c wild-type and chaperone-inactive mutants. The kinase and reporter activities were analyzed as described in (for reporter assay, n = 6, * P <0.0001 versus unstimulated counterpart). D . In vitro kinase activity of IKK in the presence of recombinant Hsp60 protein. The IKK complex was immunoprecipitated from HeLa cell lysates and incubated with or without the indicated GST proteins (20 µg each) in the kinase reaction buffer for 10 min before the kinase reaction. E . Serine phosphorylation of IKKα/β in HeLa cells transfected with AS-1 ODN. Data in the graph are means ± S.D. ( n = 3, * P <0.02, * P <0.001). F . Serine phosphorylation of IKKα/β in Hsp60c-expression HeLa cells. A representative blot is shown ( n = 3).

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Activation Assay, Expressing, Reporter Assay, In Vitro, Activity Assay, Recombinant, Immunoprecipitation, Incubation, Phospho-proteomics, Transfection

Journal: PLoS ONE

Article Title: Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

doi: 10.1371/journal.pone.0009422

Figure Lengend Snippet: A . Schematic representation of transgenic vector containing HA-tagged human Hsp60c. B . Identification of two transgenic mouse lines (T4 and T11). Genomic PCR was performed using two different sets of PCR primers, both of which were specific to Hsp60 transgene. The transgenic vector (V) and C57BL/6j (B6) mouse genomic DNA were used as positive and negative control, respectively. C . Expression of Hsp60c protein in various tissues of transgenic mice. The tissue homogenates were immunoblotted using anti-HA antibody. D . IKK activation in the liver of control B6 mice or HA-Hsp60c-expressing transgenic mice (T4 and T11) intravenously injected with TNF-α. E and F . DEN-induced cell death in the liver of control and transgenic mice primed with or without TNF-α, as measured by TUNEL assay. The representative images ( E ) are shown. The quantified data in the graph ( F ) are means ± S.E.M. of the number of TUNEL-positive cells per unit area ( n = 3, * P <0.01 versus paired stimulated one).

Article Snippet: Antibodies to IKKα (B-8), IKKγ (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IκBα (C-21), JNK1 (C-17), ASK-1 (H-300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti-Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine antibody was obtained from Qiagen; Antibodies to phosphor-IKK, IKKα, and IKKβ were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Antibodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglutinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-α was purchased from Invitrogen (Grand Island, USA).

Techniques: Transgenic Assay, Plasmid Preparation, Negative Control, Expressing, Activation Assay, Control, Injection, TUNEL Assay