c jun blocking peptide  (Tocris)

Journal: Viruses

Article Title: Regulation of Stress-Activated Kinases in Response to Tacaribe Virus Infection and Its Implications for Viral Replication

doi: 10.3390/v14092018

Figure Lengend Snippet: Host cell kinase inhibitors and activators used for growth kinetics.

Article Snippet: c-Jun ↓ , c-JUN blocking peptide , Tocris # 1989 , 50 μM.

Techniques: Blocking Assay

Journal: Cell Death & Disease

Article Title: Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

doi: 10.1038/cddis.2010.1

Figure Lengend Snippet: Galectin-1 (Gal-1)-induced phosphorylation kinetics of c-Jun and inhibition of c-Jun Ser63/73 phosphorylation with SP600125, protein kinase C-θ (PKCθ) inhibitor, and lactose ( A ) and with desipramine, imipramine, and myricetin ( B ). Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with PKCθ inhibitor for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, and with the ATP-competitive inhibitor for c-Jun N-terminal kinase (JNK) SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) with myricetin for 30 min as indicated. Control cells were incubated in medium alone. Non- and inhibitor-treated cells were then stimulated with gal-1 without and in the presence of lactose. Cell extract proteins were analyzed on blots with a phospho-c-Jun (Ser63) polyclonal antibody (pAb) and a phospho-c-Jun (Ser73) pAb without ( A , panel a; B ) and in the presence of c-Jun (Ser63) and c-Jun (Ser73) blocking peptides at 4 μ g/ml ( A , panel b). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. Shown are representative blots from three independent experiments

Article Snippet: Bad pAb, Bcl-2 pAb, phospho-Bcl-2 (Ser70) monoclonal antibody (mAb), phospho-Bcl-2 (Thr56) pAb, cleaved caspase-9 (Asp315) pAb, cleaved caspase-3 (Asp175) rabbit mAb, phospho-c-Jun (Ser63) pAb, phospho-c-Jun (Ser63) blocking peptide, phospho-c-Jun (Ser73) pAb, phospho-c-Jun (Ser73) blocking peptide, phospho-MKK3/6 (Ser189/Thr207) mAb, phospho-MKK7 (Ser271/Thr275) pAb, phospho-JNK (Thr183/Tyr185) mAb, phospho-MKK4 (Ser257/Thr261) pAb, and the JNK assay kit were from New England Biolabs (Frankfurt, Germany).

Techniques: Inhibition, Incubation, Blocking Assay, Expressing

Journal: Cell Death & Disease

Article Title: Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

doi: 10.1038/cddis.2010.1

Figure Lengend Snippet: Galectin-1 (Gal-1)-induced phosphorylation of c-Jun N-terminal kinase 1 (JNK1) and JNK2 ( a ) and JNK activation with c-Jun(1-169)-GST ( b ), and c-Jun(1-89)-GST ( c ) as kinase substrates. Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with protein kinase C-θ (PKCθ) inhibitor and PKCδ inhibitor rottlerin for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, as well as with the ATP-competitive inhibitor for JNK SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) myricetin for 30 min as indicated. Control cells were incubated in medium alone. Cells were then stimulated with gal-1 without and in the presence of lactose or asialofetuin as indicated in panels a , b , and c . ( a ) For immunoblot analysis cell extract proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were analyzed with a phospho-JNK (Thr183/Tyr185) monoclonal antibody (mAb). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. ( b ) After termination of the kinase reactions with 3 × SDS sample buffer, samples were electrophoretically separated and blotted on PVDF membranes. The [ 32 P]-labeled substrate c-Jun(1-169)-GST was recorded by autoradiography. To control loading, we separated 50 μ g cell extract protein/lane and blotted it on PVDF membranes. Membranes were probed with a JNK1 polyclonal antibody (pAb). ( c ) After termination of the kinase reactions, samples were separated and blotted on Hybond ECL membranes. Blots were analyzed for substrate phosphorylation with a phospho-c-Jun (Ser63) pAb. The bands were luminographically visualized on X-ray films using ECL Plus reagents. Shown are representative blots from three independent experiments

Article Snippet: Bad pAb, Bcl-2 pAb, phospho-Bcl-2 (Ser70) monoclonal antibody (mAb), phospho-Bcl-2 (Thr56) pAb, cleaved caspase-9 (Asp315) pAb, cleaved caspase-3 (Asp175) rabbit mAb, phospho-c-Jun (Ser63) pAb, phospho-c-Jun (Ser63) blocking peptide, phospho-c-Jun (Ser73) pAb, phospho-c-Jun (Ser73) blocking peptide, phospho-MKK3/6 (Ser189/Thr207) mAb, phospho-MKK7 (Ser271/Thr275) pAb, phospho-JNK (Thr183/Tyr185) mAb, phospho-MKK4 (Ser257/Thr261) pAb, and the JNK assay kit were from New England Biolabs (Frankfurt, Germany).

Techniques: Activation Assay, Incubation, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page, Expressing, Labeling, Autoradiography

Journal: Cell Death & Disease

Article Title: Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

doi: 10.1038/cddis.2010.1

Figure Lengend Snippet: Galectin-1 (Gal-1)-induced phosphorylation kinetics of c-Jun and inhibition of c-Jun Ser63/73 phosphorylation with SP600125, protein kinase C-θ (PKCθ) inhibitor, and lactose ( A ) and with desipramine, imipramine, and myricetin ( B ). Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with PKCθ inhibitor for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, and with the ATP-competitive inhibitor for c-Jun N-terminal kinase (JNK) SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) with myricetin for 30 min as indicated. Control cells were incubated in medium alone. Non- and inhibitor-treated cells were then stimulated with gal-1 without and in the presence of lactose. Cell extract proteins were analyzed on blots with a phospho-c-Jun (Ser63) polyclonal antibody (pAb) and a phospho-c-Jun (Ser73) pAb without ( A , panel a; B ) and in the presence of c-Jun (Ser63) and c-Jun (Ser73) blocking peptides at 4 μ g/ml ( A , panel b). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. Shown are representative blots from three independent experiments

Article Snippet: Bad pAb, Bcl-2 pAb, phospho-Bcl-2 (Ser70) monoclonal antibody (mAb), phospho-Bcl-2 (Thr56) pAb, cleaved caspase-9 (Asp315) pAb, cleaved caspase-3 (Asp175) rabbit mAb, phospho-c-Jun (Ser63) pAb, phospho-c-Jun (Ser63) blocking peptide, phospho-c-Jun (Ser73) pAb, phospho-c-Jun (Ser73) blocking peptide, phospho-MKK3/6 (Ser189/Thr207) mAb, phospho-MKK7 (Ser271/Thr275) pAb, phospho-JNK (Thr183/Tyr185) mAb, phospho-MKK4 (Ser257/Thr261) pAb, and the JNK assay kit were from New England Biolabs (Frankfurt, Germany).

Techniques: Inhibition, Incubation, Blocking Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: Synaptogenesis Regulates Axotomy-Induced Activation of c-Jun–Activator Protein-1 Transcription

doi: 10.1523/JNEUROSCI.1844-06.2006

Figure Lengend Snippet: Characterizing the injury-induced AP1 protein complex. A, Antibodies to p73–c-Jun, p-ATF-2, c-Jun(N), c-Jun(D), Rel, c-Fos, and JunD were preincubated with pleural ganglion neuronal extracts (obtained 1 d after nerve crush) and binding to the 32P-labeled TRE oligonucleotide was assayed via EMSA. An asterisk denotes supershifted complexes. B, Supershift assay at the indicated times after nerve crush using p73–c-Jun and c-Jun(N) antibodies as in A. Neurons from four animals were combined for all of the supershift assays presented in A and B. C, The level of p73–c-Jun is elevated in sensory neuron nuclei after nerve crush. At 2 d and 14 d after nerve crush, the sensory neuron cluster from the injured (I-2d and I-14d) and contralateral control sides (CC-2d and CC-14d) were removed and immunostained with Ab-p73-cJun. Two micrometer optical sections showing the nucleus were obtained by confocal microscopy. Staining of uninjured (Naive) neurons, background staining of a 2 d injured sample in the absence of primary antibody (second Ab), and the staining a 2 d injured sample in the presence of a p73–c-Jun blocking peptide are also shown. Scale bar, 50 μm.

Article Snippet: Antibodies to phosphorylated JNK (p-JNK) (recognizes the dually phosphorylated form of JNK1/2/3), Ser-73 (p73–c-Jun) and its blocking peptide, and an antibody to ATF-2 phosphorylated on Thr-69/71 (p-ATF-2) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Binding Assay, Labeling, Confocal Microscopy, Staining, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Synaptogenesis Regulates Axotomy-Induced Activation of c-Jun–Activator Protein-1 Transcription

doi: 10.1523/JNEUROSCI.1844-06.2006

Figure Lengend Snippet: A, PHYLIP analysis of the JNK family. Sequence identities of Aedes albopictus AaJNK (GenBank accession number AAO31950), Ciona intestinalis CiJNK (GenBank accession number BAE06525), Hydra vulgaris HvJNK (GenBank accession number AAU93351), Caenorhabditis elegans CeJNK1 (GenBank accession number NP_741434), Suberites domuncula SdJNK (GenBank accession number CAC85496), Drosophila dJNK (GenBank accession number AAC47325) and Basket (GenBank accession number P92208), human hJNK1 (GenBank accession number NP_620637), hJNK2 (GenBank accession number NP_620707) and hJNK3 (GenBank accession number P53779), rat rJNK1 (GenBank accession number P49185), rJNK2 (GenBank accession number P49186), and rJNK3 (GenBank accession number P49187), mouse mJNK1 (GenBank accession number Q91Y86), mJNK2 (GenBank accession number Q9WTU6), and mJNK3 (GenBank accession number Q61831), and Aplysia californica apMAPK (GenBank accession number AAA83210) were computed with respect to apJNK by CLUSTAL-W and PHYLIP (version 3.6) (http://workbench.sdsc.edu/); percentages are shown in parentheses. B, p-JNK antibody recognizes apJNK in neuronal lysates. Western blot of 20 μg of protein extracted from contralateral uninjured (lane1) and injured (lane 2) pleural neurons 1 d after nerve crush was probed with Ab-p-JNK. C, Expression and purification of apJNK. Lane 1, Autoradiograph after SDS-PAGE showing the expression of 35S-labeled recombinant His–apJNK–GFP produced by in vitro translation. Lane 2, The recombinant protein after affinity purification. D, Recombinant His–apJNK–GFP phosphorylates a recombinant GST–c-Jun fusion protein containing the Ser-73 site. His–apJNK–GFP activity was assayed by the transfer of 32P from [γ-32P]ATP to GST-c-Jun(1–79) in vitro, followed by autoradiography. E, Recombinant His–apJNK–GFP phosphorylates endogenous c-Jun at Ser-73 in vitro. Purified His–apJNK–GFP was added to an extract prepared from uninjured neurons, and the production of c-Jun phosphorylated at Ser-73 was assessed by probing a Western blot with Ab-p73–c-Jun.

Article Snippet: Antibodies to phosphorylated JNK (p-JNK) (recognizes the dually phosphorylated form of JNK1/2/3), Ser-73 (p73–c-Jun) and its blocking peptide, and an antibody to ATF-2 phosphorylated on Thr-69/71 (p-ATF-2) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Sequencing, Western Blot, Expressing, Purification, Autoradiography, SDS Page, Labeling, Recombinant, Produced, In Vitro, Affinity Purification, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Synaptogenesis Regulates Axotomy-Induced Activation of c-Jun–Activator Protein-1 Transcription

doi: 10.1523/JNEUROSCI.1844-06.2006

Figure Lengend Snippet: Cultured neurons express p73–c-Jun and AP1 transcriptional activity. A, Representative confocal images from two experiments showing Ab-p-JNK (green, left) and Ab-p73–c-Jun (red, middle) immunofluorescence in 2 μm optical sections through the nucleus of the sensory neurons on day 2 in vitro. B, AP1-dependent expression of a heterologous reporter protein (GFP) in injured cultured sensory neurons. pNEX–GFP or pNEXAP1–GFP plasmid DNA was injected into sensory neurons on day 1 in vitro (5 sensory neurons per plasmid). GFP expression was examined 16 h later by fluorescence microscopy. Fluorescence was only observed in pNEXAP1–GFP injected cells (20% expression; n = 3 experiments). C, Nuclear translocation of apJNK after axotomy in vitro. Representative images from two experiments showing p-apJNK immunofluorescence in the nucleus of sensory neurons after 7 d in vitro. The top row shows 2 μm optical sections of control sensory neurons, and the bottom row shows sensory neurons that were axotomized 15 min before fixation. D, SP600125 inhibits the ability of apJNK to phosphorylate c-Jun. A total of 5 μg of injured pleural ganglia extract was incubated with GST–c-Jun(1–79) or Elk1 in the presence or absence of SP600125 at the indicated concentrations. apJNK activity was assayed by the transfer of 32P from [γ-32P]ATP to GST–c-Jun(1–79) or Elk1 in vitro, followed by autoradiography. E, Inhibiting axotomy-induced apJNK activation reduces the phosphorylation of c-Jun. The 4 d sensory neurons were axotomized in the presence of 1 μm SP600125 or its nonactive form, NmP, and were placed in vitro in the continued presence of the drugs. At 1 d later, the cells were fixed and exposed to Ab-p73–c-Jun to detect p73–c-Jun in the nucleus.

Article Snippet: Antibodies to phosphorylated JNK (p-JNK) (recognizes the dually phosphorylated form of JNK1/2/3), Ser-73 (p73–c-Jun) and its blocking peptide, and an antibody to ATF-2 phosphorylated on Thr-69/71 (p-ATF-2) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Cell Culture, Activity Assay, Immunofluorescence, In Vitro, Expressing, Plasmid Preparation, Injection, Fluorescence, Microscopy, Translocation Assay, Incubation, Autoradiography, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Synaptogenesis Regulates Axotomy-Induced Activation of c-Jun–Activator Protein-1 Transcription

doi: 10.1523/JNEUROSCI.1844-06.2006

Figure Lengend Snippet: A, Photomicrographs of 4 d sensory neuron (SN)–L7 (left) and SN–L11 (right) cocultures near the contact point between the major axons and the sensory neuron arbor. Individual cells marked by colored arrows: SN, black; L7–L11, blue. Scale bar, 50 μm. B, Decrease in p73–c-Jun and p-apJNK activity in sensory neurons after contact with L7. SN–SN, SN–L7, and SN–L11 cocultures were fixed on day 4 and then immunostained for p73–c-Jun (red, left) and p-apJNK (green, middle). Top five rows, Representative examples of SN–SN, SN–L7, and SN–L11 cultures. Bottom row, Secondary antibody control. Each image is a 2 μm optical section through the sensory cell body to show the nucleus. Scale bar, 50 μm. C, Histogram of the Mean ± SEM staining intensity of SN nuclear p73–c-Jun and p-apJNK immunoreactivity from three separate experiments. The number of cells in the SN (black bar), SN–L7 (blank bar), and SN–L11 (gray bar) groups are 12, 10, and 8, respectively. The asterisks indicate that the SN–L7 data are significantly different from sensory neurons alone and SN–L11 cocultures as described in Results.

Article Snippet: Antibodies to phosphorylated JNK (p-JNK) (recognizes the dually phosphorylated form of JNK1/2/3), Ser-73 (p73–c-Jun) and its blocking peptide, and an antibody to ATF-2 phosphorylated on Thr-69/71 (p-ATF-2) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Synaptogenesis Regulates Axotomy-Induced Activation of c-Jun–Activator Protein-1 Transcription

doi: 10.1523/JNEUROSCI.1844-06.2006

Figure Lengend Snippet: Inhibiting JNK activity increases synaptic activity. L7 and sensory neuron were cocultured in the presence of 1 μm SP600125 or NmP. EPSPs were recorded on day 2. A, Top, Representative EPSPs recorded in L7. SP600125 treatment increased the amplitude of EPSPs when compared with NmP treatment. Calibration: 25 ms, 25 mV. Bottom, Summary of the effect of SP600125 on EPSP amplitudes. Error bars indicate SEM; *p < 0.0001. B, Representative examples of p73–c-Jun expression in cocultured sensory neuron (SN) and L7 in the presence of NmP or SP600125 from three separate experiments. SN–L7 cocultures were fixed immediately after EPSP recording and then immunostained for p73–c-Jun. Each image is a 2 μm optical section through the sensory cell body to show the nucleus. Scale bar, 20 μm. The histogram shows the ratio of nuclear p73–c-Jun staining intensity/EPSP in SN cells treated with NmP (white bar) or SP600125 (black bars). A total of 20 SNs were assessed under each condition (p < 0.0035, Scheffé’s test).

Article Snippet: Antibodies to phosphorylated JNK (p-JNK) (recognizes the dually phosphorylated form of JNK1/2/3), Ser-73 (p73–c-Jun) and its blocking peptide, and an antibody to ATF-2 phosphorylated on Thr-69/71 (p-ATF-2) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay, Expressing, Staining