Structured Review

Santa Cruz Biotechnology anti c jun
AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in <t>anti-c-Jun</t> antibody-immunoprecipited candidates, but not in <t>anti-IgG</t> antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P
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1) Product Images from "The histidine-rich calcium binding protein (HRC) promotes tumor metastasis in hepatocellular carcinoma and is upregulated by SATB1"

Article Title: The histidine-rich calcium binding protein (HRC) promotes tumor metastasis in hepatocellular carcinoma and is upregulated by SATB1

Journal: Oncotarget

doi:

AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in anti-c-Jun antibody-immunoprecipited candidates, but not in anti-IgG antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P
Figure Legend Snippet: AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in anti-c-Jun antibody-immunoprecipited candidates, but not in anti-IgG antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P

Techniques Used: Expressing, Luciferase, Activity Assay, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Mutagenesis, Polymerase Chain Reaction, Activation Assay

2) Product Images from "Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1"

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.14.5933-5946.2005

Effect of TBH on electrophoretic mobility shift and supershift assays for AP-1 binding. Nuclear protein extracts (10 μg) were obtained from WT, F1, and F2 cells treated with TBH (60 μM for 0, 4, or 8 h), and EMSA was done as described in Materials and Methods using a consensus AP-1 probe. Panel A shows supershift analysis using anti-c-Jun antibodies, and panel B shows supershift analysis using anti-c-Fos antibodies. The arrows to the right point to complexes that were supershifted in the presence of specific antibodies. Representative EMSAs are shown.
Figure Legend Snippet: Effect of TBH on electrophoretic mobility shift and supershift assays for AP-1 binding. Nuclear protein extracts (10 μg) were obtained from WT, F1, and F2 cells treated with TBH (60 μM for 0, 4, or 8 h), and EMSA was done as described in Materials and Methods using a consensus AP-1 probe. Panel A shows supershift analysis using anti-c-Jun antibodies, and panel B shows supershift analysis using anti-c-Fos antibodies. The arrows to the right point to complexes that were supershifted in the presence of specific antibodies. Representative EMSAs are shown.

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

EMSA and supershift analysis of the rat GCLC AP-1 and NF-κB sites. WT cells were treated with TBH (60 μM for 8 h) or vehicle control and subjected to EMSA with supershift analysis for the AP-1 site at −356 or the NF-κB site at −378. Supershift analysis was performed using antibodies directed against c-Jun, Nrf1, and Nrf2 for the AP-1 site (A) and p50, Nrf1, and Nrf2 for the NF-κB site (B). Note that supershift occurred only with anti-c-Jun antibodies for the AP-1 site and anti-p50 antibodies for the NF-κB site. As a positive control, TBH treatment induced Nrf1 and Nrf2 binding to the ARE site of the mouse GCLM (C). Arrows in panel C point to the Nrf1 and Nrf2 supershifts.
Figure Legend Snippet: EMSA and supershift analysis of the rat GCLC AP-1 and NF-κB sites. WT cells were treated with TBH (60 μM for 8 h) or vehicle control and subjected to EMSA with supershift analysis for the AP-1 site at −356 or the NF-κB site at −378. Supershift analysis was performed using antibodies directed against c-Jun, Nrf1, and Nrf2 for the AP-1 site (A) and p50, Nrf1, and Nrf2 for the NF-κB site (B). Note that supershift occurred only with anti-c-Jun antibodies for the AP-1 site and anti-p50 antibodies for the NF-κB site. As a positive control, TBH treatment induced Nrf1 and Nrf2 binding to the ARE site of the mouse GCLM (C). Arrows in panel C point to the Nrf1 and Nrf2 supershifts.

Techniques Used: Positive Control, Binding Assay

Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P
Figure Legend Snippet: Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P

Techniques Used: Western Blot

3) Product Images from "Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity"

Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity

Journal: Molecular Endocrinology

doi: 10.1210/me.2008-0211

Differential association of AP-1 components and ERβ with the PTPRO promoter in the presence of E2 and tamoxifen. A, Whole-cell extracts from Hs578t, 48R, and MCF-7 cells were subjected to Western blot analysis with anti-c-Fos and anti-c-Jun antibody.
Figure Legend Snippet: Differential association of AP-1 components and ERβ with the PTPRO promoter in the presence of E2 and tamoxifen. A, Whole-cell extracts from Hs578t, 48R, and MCF-7 cells were subjected to Western blot analysis with anti-c-Fos and anti-c-Jun antibody.

Techniques Used: Western Blot

4) Product Images from "Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase"

Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Effect of EGF on the interaction between Sp1 oligonucleotides and c-Jun/Sp1. Nuclear extracts from EGF-treated cells were prepared and subjected to the assay for binding of the c-Jun/Sp1 complex to Sp1 consensus sites. The 32 P-radiolabeled Sp1 oligonucleotide (●) and Sp1 mutant SPM (□) were used as a probe for binding, respectively. For background control, protein A-agarose was used to substitute anti-c-Jun antibody–agarose conjugate in the assay (■). Values are means ± SEM of three determinations.
Figure Legend Snippet: Effect of EGF on the interaction between Sp1 oligonucleotides and c-Jun/Sp1. Nuclear extracts from EGF-treated cells were prepared and subjected to the assay for binding of the c-Jun/Sp1 complex to Sp1 consensus sites. The 32 P-radiolabeled Sp1 oligonucleotide (●) and Sp1 mutant SPM (□) were used as a probe for binding, respectively. For background control, protein A-agarose was used to substitute anti-c-Jun antibody–agarose conjugate in the assay (■). Values are means ± SEM of three determinations.

Techniques Used: Binding Assay, Mutagenesis

Binding of c-Jun and Sp1 in cells overexpressing c-Jun or Ha-ras. Cells were transfected with a different amount of pRSVjun or pSV2ras by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun protein ( A ) and the coimmunoprecipitated c-Jun/Sp1 complex by using anti-Sp1 antibodies ( B and C ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies.
Figure Legend Snippet: Binding of c-Jun and Sp1 in cells overexpressing c-Jun or Ha-ras. Cells were transfected with a different amount of pRSVjun or pSV2ras by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun protein ( A ) and the coimmunoprecipitated c-Jun/Sp1 complex by using anti-Sp1 antibodies ( B and C ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies.

Techniques Used: Binding Assay, Transfection, Incubation, Expressing, Western Blot

Effect of c-Jun dominant negative mutant on c-Jun/Sp1 interaction in pRSVjun-treated cells. Cells were transfected with 2 μg of pRSVjun and a different amount of c-Jun dominant negative vector TAM-67 by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun and TAM-67 proteins ( A ) and the coimmunoprecipitated c-Jun/Sp1 and TAM-67/Sp1 complex by using anti-Sp1 antibodies ( B ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies. pcDNA3.1 was used as a vector to adjust for the same amount of plasmids in each experiment.
Figure Legend Snippet: Effect of c-Jun dominant negative mutant on c-Jun/Sp1 interaction in pRSVjun-treated cells. Cells were transfected with 2 μg of pRSVjun and a different amount of c-Jun dominant negative vector TAM-67 by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun and TAM-67 proteins ( A ) and the coimmunoprecipitated c-Jun/Sp1 and TAM-67/Sp1 complex by using anti-Sp1 antibodies ( B ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies. pcDNA3.1 was used as a vector to adjust for the same amount of plasmids in each experiment.

Techniques Used: Dominant Negative Mutation, Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot

5) Product Images from "The MicroRNA-130/301 Family Controls Vasoconstriction in Pulmonary Hypertension *"

Article Title: The MicroRNA-130/301 Family Controls Vasoconstriction in Pulmonary Hypertension *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.617845

The miR-130/301 family controls the transcription of endothelin-1 through a specific PPARγ-AP1 (c-Fos/c-Jun) axis. A , in PAECs, RT-qPCR revealed that miR-130a-dependent alterations in EDN1, but not VEGFA, were reversed by siRNA knockdown of c-Jun
Figure Legend Snippet: The miR-130/301 family controls the transcription of endothelin-1 through a specific PPARγ-AP1 (c-Fos/c-Jun) axis. A , in PAECs, RT-qPCR revealed that miR-130a-dependent alterations in EDN1, but not VEGFA, were reversed by siRNA knockdown of c-Jun

Techniques Used: Quantitative RT-PCR

6) Product Images from "The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation"

Article Title: The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2018.09.002

MDX induces IRE1β expression via P38 MAPK.  ( A ) MDX induces  Ern-2  expression in the HT29-MTX cell line. Cells were either left untreated (UT) or cultured with increasing concentrations of MDX for 1 hour.  Ern-2  RNA transcripts were assessed by real-time PCR. Data are means ± SD of 10 samples per group derived from 4 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (** P  ≤ .01, * P  ≤ .05). ( B ) HT29-MTX cells were either left UT or stimulated with 5% MDX for the indicated time points. p-p38, p38, phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (β-act) expression were analyzed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P  ≤ .05, ** P  ≤ .01, *** P  ≤ .001). ( C ) Effect of the p38 inhibitor (p38i) on MDX-mediated p38 activation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or dimethyl sulfoxide (DMSO) (vehicle). p-p38 and p38 expression was assessed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with the densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*** P  ≤ .001).  Right : Effect of p38i on MDX-mediated  Ern-2  up-regulation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or DMSO (vehicle) and  Ern-2  RNA transcripts evaluated by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P  ≤ .05). ( D ) Effect of p38 knock-down on MDX-mediated  Ern-2  up-regulation. HT29-MTX cells were transfected with either control or p38 siRNA (CTR or p38 siRNA, respectively) for 18 hours and then stimulated or not with 5% MDX for 1 hour. Representative Western blot for p-p38, p38, and β-actin expression are shown in the  left panels  together with the densitometry analysis. Data are means ± SD of 3 independent experiments. Differences between groups were compared using the 2-tailed Student  t  test (** P  ≤ .001).  Right : Expression of  Ern-2  RNA transcripts assessed by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P  ≤ .05). ( E ) Immunofluorescence analysis of p-p38 (green) in colon samples isolated from MDX-fed mice and controls killed on day 45. The figure is representative of 3 separate experiments in which similar results were obtained.  Right panels  show the number and intensity of p-p38–expressing cells per field of colon section. Data are expressed as means ± SD and were generated using 3–5 mice per group from 3 independent experiments. Differences between groups were compared using the 2-tailed Student  t  test (** P  ≤ .01). mRNA, messenger RNA; p38 tot, total p38.
Figure Legend Snippet: MDX induces IRE1β expression via P38 MAPK. ( A ) MDX induces Ern-2 expression in the HT29-MTX cell line. Cells were either left untreated (UT) or cultured with increasing concentrations of MDX for 1 hour. Ern-2 RNA transcripts were assessed by real-time PCR. Data are means ± SD of 10 samples per group derived from 4 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (** P ≤ .01, * P ≤ .05). ( B ) HT29-MTX cells were either left UT or stimulated with 5% MDX for the indicated time points. p-p38, p38, phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (β-act) expression were analyzed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05, ** P ≤ .01, *** P ≤ .001). ( C ) Effect of the p38 inhibitor (p38i) on MDX-mediated p38 activation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or dimethyl sulfoxide (DMSO) (vehicle). p-p38 and p38 expression was assessed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with the densitometry analysis ( lower panel ). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*** P ≤ .001). Right : Effect of p38i on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or DMSO (vehicle) and Ern-2 RNA transcripts evaluated by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). ( D ) Effect of p38 knock-down on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were transfected with either control or p38 siRNA (CTR or p38 siRNA, respectively) for 18 hours and then stimulated or not with 5% MDX for 1 hour. Representative Western blot for p-p38, p38, and β-actin expression are shown in the left panels together with the densitometry analysis. Data are means ± SD of 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (** P ≤ .001). Right : Expression of Ern-2 RNA transcripts assessed by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (* P ≤ .05). ( E ) Immunofluorescence analysis of p-p38 (green) in colon samples isolated from MDX-fed mice and controls killed on day 45. The figure is representative of 3 separate experiments in which similar results were obtained. Right panels show the number and intensity of p-p38–expressing cells per field of colon section. Data are expressed as means ± SD and were generated using 3–5 mice per group from 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (** P ≤ .01). mRNA, messenger RNA; p38 tot, total p38.

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Derivative Assay, Activated Clotting Time Assay, Western Blot, Activation Assay, Transfection, Immunofluorescence, Isolation, Mouse Assay, Generated

7) Product Images from "Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves' Disease: Apparent Role of ADAM17"

Article Title: Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves' Disease: Apparent Role of ADAM17

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034173

IGF-1Rβ phosphorylation may play an essential role in the nuclear translocation of IGF-1Rα in GD orbital fibroblasts. ( A ) NVP-AEW-541, a specific tyrosine kinase inhibitor blocks IGF-1R phosphorylation ( B ) NVP-AEW-541 blocks nuclear IGF-1Rα accumulation. GD orbital fibroblasts were treated as indicated and stained with anti-IGF-1Rα Ab (green) and counterstained with PI. ( C ) Western blot analysis of nuclear proteins from GD orbital fibroblasts treated with nothing, IGF-1, NVP-AEW-541, or the combination and probed with anti- IGF-1Rα and anti-c-jun Abs. The findings are representative of three experiments performed.
Figure Legend Snippet: IGF-1Rβ phosphorylation may play an essential role in the nuclear translocation of IGF-1Rα in GD orbital fibroblasts. ( A ) NVP-AEW-541, a specific tyrosine kinase inhibitor blocks IGF-1R phosphorylation ( B ) NVP-AEW-541 blocks nuclear IGF-1Rα accumulation. GD orbital fibroblasts were treated as indicated and stained with anti-IGF-1Rα Ab (green) and counterstained with PI. ( C ) Western blot analysis of nuclear proteins from GD orbital fibroblasts treated with nothing, IGF-1, NVP-AEW-541, or the combination and probed with anti- IGF-1Rα and anti-c-jun Abs. The findings are representative of three experiments performed.

Techniques Used: Translocation Assay, Staining, Western Blot

IGF-1R protein differentially accumulates in the nuclei of TAO orbital fibroblasts and derives from the fibroblast surface. ( A ) Western blot analysis of nuclear and cytoplasmic IGF-1Rα in GD orbital fibroblasts before and following IGF-1 (10 nM) treatment for 16 h. Cells were subjected to subcellular fractionation as described in “ Methods ” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. ( B ) Nuclear IGF-1Rα content in GD and control orbital fibroblasts before or following treatment with either IGF-1 (10 nM) or GD-IgG (15 µg/ml) for 16 hours. ( C ) Insulin fails to alter the nuclear content of IR or IGF-1Rα in GD orbital fibroblasts. Cells were treated with nothing or insulin (15 µg/ml) for 16 hrs. They were subjected to subcellular fractionation and Western blot analysis. ( D ) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. ( E ) Control and GD fibroblasts were subjected to 125 I-IGF-1 cross-linking with either the cell-impermeable agent, BS, or the cell permeable agent, DSS. They were then treated with IGF-1. Nuclei were separated as described in “ Methods ” and subjected to quantification of radioactivity. Results are representative of three experiments performed.
Figure Legend Snippet: IGF-1R protein differentially accumulates in the nuclei of TAO orbital fibroblasts and derives from the fibroblast surface. ( A ) Western blot analysis of nuclear and cytoplasmic IGF-1Rα in GD orbital fibroblasts before and following IGF-1 (10 nM) treatment for 16 h. Cells were subjected to subcellular fractionation as described in “ Methods ” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. ( B ) Nuclear IGF-1Rα content in GD and control orbital fibroblasts before or following treatment with either IGF-1 (10 nM) or GD-IgG (15 µg/ml) for 16 hours. ( C ) Insulin fails to alter the nuclear content of IR or IGF-1Rα in GD orbital fibroblasts. Cells were treated with nothing or insulin (15 µg/ml) for 16 hrs. They were subjected to subcellular fractionation and Western blot analysis. ( D ) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. ( E ) Control and GD fibroblasts were subjected to 125 I-IGF-1 cross-linking with either the cell-impermeable agent, BS, or the cell permeable agent, DSS. They were then treated with IGF-1. Nuclei were separated as described in “ Methods ” and subjected to quantification of radioactivity. Results are representative of three experiments performed.

Techniques Used: Western Blot, Fractionation, Radioactivity

8) Product Images from "Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure"

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.38841

Knockdown or blockage of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . (A) Knockdown of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . The RPTEC/TERT1 OAT3 cells were transfected with two independent siRNAs of CtBP2 . After incubation for another 48 h, cells were further treated with or without 50 ng/mL IL-1β for 2 h, followed by ChIP assays using anti-CtBP2, anti-p300, anti-c-Jun and anti-c-FOS to determine their occupancies in the promoter of TGFB1 . ** P
Figure Legend Snippet: Knockdown or blockage of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . (A) Knockdown of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . The RPTEC/TERT1 OAT3 cells were transfected with two independent siRNAs of CtBP2 . After incubation for another 48 h, cells were further treated with or without 50 ng/mL IL-1β for 2 h, followed by ChIP assays using anti-CtBP2, anti-p300, anti-c-Jun and anti-c-FOS to determine their occupancies in the promoter of TGFB1 . ** P

Techniques Used: Transfection, Incubation, Chromatin Immunoprecipitation

9) Product Images from "Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling"

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling

Journal: International Journal of Oncology

doi: 10.3892/ijo.2013.2079

Effects of Cf-GP on the expression levels of Tcf, LEF-1, ICAM-1, c-jun, c-myc and cyclin D. Cells were treated with Cf-GP (5, 10 or 20 μ g/ml) for 24 h. Gene expression was determined by western blot analysis and RT-PCR. (A) Western blot analysis using anti-Tcf, anti-LEF-1, anti-ICAM-1, anti-c-jun, anti-c-myc, anti-cyclin D and anti-β-actin antibodies. SDS-PAGE was performed on acrylamide gel. (B) cDNA and primers were synthesized. PCR was then performed at the indicated annealing temperatures. Reaction products were electrophoresed on a 1% agarose gel and visualized with RedSafe reagent.
Figure Legend Snippet: Effects of Cf-GP on the expression levels of Tcf, LEF-1, ICAM-1, c-jun, c-myc and cyclin D. Cells were treated with Cf-GP (5, 10 or 20 μ g/ml) for 24 h. Gene expression was determined by western blot analysis and RT-PCR. (A) Western blot analysis using anti-Tcf, anti-LEF-1, anti-ICAM-1, anti-c-jun, anti-c-myc, anti-cyclin D and anti-β-actin antibodies. SDS-PAGE was performed on acrylamide gel. (B) cDNA and primers were synthesized. PCR was then performed at the indicated annealing temperatures. Reaction products were electrophoresed on a 1% agarose gel and visualized with RedSafe reagent.

Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, SDS Page, Acrylamide Gel Assay, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis

10) Product Images from "EBV-LMP1 targeted DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via the JNKs/HIF-1 pathway in nasopharyngeal carcinoma"

Article Title: EBV-LMP1 targeted DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via the JNKs/HIF-1 pathway in nasopharyngeal carcinoma

Journal: Oncotarget

doi:

The JNKs/c-Jun signaling pathway is involved in LMP1-induced HIF-1 and VEGF expression Cells were treated with DZ1 or CDN and 400 μM CoCl 2  for 24 h. (A,B) Western blotting was performed to analyze the expression of LMP1 and its downstream targets, including phosphorylated JNKs, JNKs, c-Jun, phosphorylated c-Jun, HIF-1 and VEGF in CNE1/CNE1-LMP1 cells and HNE2/HNE21-LMP1 cells. (C, D) HIF-1 activity levels were examined in these cells using the  HRE  reporter gene assay. Data were expressed as the means ± S.D. of three experiments. (E, F, G, H) Quantitative RT-PCR was used to analyze the mRNA expression of  LMP1  and  HIF-1  in CNE1/CNE1-LMP1 cells and HNE2/HNE2-LMP1 cells. The asterisk (* or **) indicates a significant (p
Figure Legend Snippet: The JNKs/c-Jun signaling pathway is involved in LMP1-induced HIF-1 and VEGF expression Cells were treated with DZ1 or CDN and 400 μM CoCl 2 for 24 h. (A,B) Western blotting was performed to analyze the expression of LMP1 and its downstream targets, including phosphorylated JNKs, JNKs, c-Jun, phosphorylated c-Jun, HIF-1 and VEGF in CNE1/CNE1-LMP1 cells and HNE2/HNE21-LMP1 cells. (C, D) HIF-1 activity levels were examined in these cells using the HRE reporter gene assay. Data were expressed as the means ± S.D. of three experiments. (E, F, G, H) Quantitative RT-PCR was used to analyze the mRNA expression of LMP1 and HIF-1 in CNE1/CNE1-LMP1 cells and HNE2/HNE2-LMP1 cells. The asterisk (* or **) indicates a significant (p

Techniques Used: Expressing, Western Blot, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR

11) Product Images from "Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts"

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138423

Involvement of activation of transcription factors, NF-κB and /AP-1, in IL-1β-induced IL-36γ expression. (A) IL-1β induced IκBα phosphorylation and degradation in colonic myofibroblasts. The cells were stimulated with IL-1β (10 ng/ml), and phosphorylated IκBα were detected by Western blotting. (B) IL-1β induced activation of NF-κB and c-Jun AP-1. The cells were stimulated with IL-1β (10 ng/ml), and NF-κB p65 and phosphorylated c-Jun were detected by immunocytochemistory. Reacted antibodies against NF-κB p65 were visualized by FITC (green fluorescence)-labeled second antibody. Reacted antibodies against phosphorylated c-Jun were detected by a DyLight ®  594 (red fluorescence)-labeled secondary antibodies. Nucleus was stained by DAPI (blue). (C) The cells were stimulated with IL-1β (10 ng/ml) or medium alone for 15 min and nuclear proteins were extracted. NF-κB p65 and phosphorylated c-Jun in nuclear extracts were detected by immunoblot. (D) Effects of silencing of NF-κB p65 and c-Jun AP-1on IL-1β-induced IL-36γ expression. The cells were transfected with control siRNA, the siRNA specific for NF-κB p65 and/or c-Jun AP-1, and incubated for 24h. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments). *P
Figure Legend Snippet: Involvement of activation of transcription factors, NF-κB and /AP-1, in IL-1β-induced IL-36γ expression. (A) IL-1β induced IκBα phosphorylation and degradation in colonic myofibroblasts. The cells were stimulated with IL-1β (10 ng/ml), and phosphorylated IκBα were detected by Western blotting. (B) IL-1β induced activation of NF-κB and c-Jun AP-1. The cells were stimulated with IL-1β (10 ng/ml), and NF-κB p65 and phosphorylated c-Jun were detected by immunocytochemistory. Reacted antibodies against NF-κB p65 were visualized by FITC (green fluorescence)-labeled second antibody. Reacted antibodies against phosphorylated c-Jun were detected by a DyLight ® 594 (red fluorescence)-labeled secondary antibodies. Nucleus was stained by DAPI (blue). (C) The cells were stimulated with IL-1β (10 ng/ml) or medium alone for 15 min and nuclear proteins were extracted. NF-κB p65 and phosphorylated c-Jun in nuclear extracts were detected by immunoblot. (D) Effects of silencing of NF-κB p65 and c-Jun AP-1on IL-1β-induced IL-36γ expression. The cells were transfected with control siRNA, the siRNA specific for NF-κB p65 and/or c-Jun AP-1, and incubated for 24h. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments). *P

Techniques Used: Activation Assay, Expressing, Western Blot, Fluorescence, Labeling, Staining, Transfection, Incubation, Real-time Polymerase Chain Reaction

12) Product Images from "Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner"

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner

Journal: Molecular and Cellular Biology

doi:

ILK-induced AP-1 activity is mediated by c-jun. (A) In this band shift assay, serum-exposed HEK-293 cells were transfected by Lipofectin with 0.5 μg of each of the indicated cDNAs. PD98059 (25 μM) was added to the medium 12 h prior to harvesting the cells. Nuclear extracts (2 μg) from the transfected cells were incubated with 32 P-end-labeled AP-1 consensus oligonucleotide containing the protein binding site. Reaction products were analyzed on a nondenaturing 5% polyacrylamide gel (left gel). The specificity of complex formation was established by a competition experiment using cold AP-1 oligonucleotide as the competitor (right gel). For immunoblot studies, 10 μg of protein was resolved by SDS–10% PAGE. c-jun (Ser-73) phosphorylation, c-jun protein expression level, ERK phosphorylation (Tyr-204), and ERK protein expression level (not shown) were determined by Western blot analysis. ILK and GSK-3 expression levels in the transfected cells were evaluated by Western blot analysis using anti-V5 and anti-HA antibodies, respectively. (B) Anti-c-jun antibody shifts ILK-induced AP-1 complex. Nuclear extracts (2 μg) from HEK-293 cells transfected with ILK-V5 cDNA were incubated with 32 P-AP-1 oligonucleotide in the absence or presence of anti-c-jun antibody or nonspecific IgG (10 μg). The latter did not induce a mobility shift of the complex (not shown).
Figure Legend Snippet: ILK-induced AP-1 activity is mediated by c-jun. (A) In this band shift assay, serum-exposed HEK-293 cells were transfected by Lipofectin with 0.5 μg of each of the indicated cDNAs. PD98059 (25 μM) was added to the medium 12 h prior to harvesting the cells. Nuclear extracts (2 μg) from the transfected cells were incubated with 32 P-end-labeled AP-1 consensus oligonucleotide containing the protein binding site. Reaction products were analyzed on a nondenaturing 5% polyacrylamide gel (left gel). The specificity of complex formation was established by a competition experiment using cold AP-1 oligonucleotide as the competitor (right gel). For immunoblot studies, 10 μg of protein was resolved by SDS–10% PAGE. c-jun (Ser-73) phosphorylation, c-jun protein expression level, ERK phosphorylation (Tyr-204), and ERK protein expression level (not shown) were determined by Western blot analysis. ILK and GSK-3 expression levels in the transfected cells were evaluated by Western blot analysis using anti-V5 and anti-HA antibodies, respectively. (B) Anti-c-jun antibody shifts ILK-induced AP-1 complex. Nuclear extracts (2 μg) from HEK-293 cells transfected with ILK-V5 cDNA were incubated with 32 P-AP-1 oligonucleotide in the absence or presence of anti-c-jun antibody or nonspecific IgG (10 μg). The latter did not induce a mobility shift of the complex (not shown).

Techniques Used: Activity Assay, Electrophoretic Mobility Shift Assay, Transfection, Incubation, Labeling, Protein Binding, Polyacrylamide Gel Electrophoresis, Expressing, Western Blot, Mobility Shift

13) Product Images from "Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer"

Article Title: Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.20160

C-Jun was required for TNC-induced MMP9 expression (A) The sequence and position of the c-Jun binding site in the MMP9 promoter are shown. (B) ChIP of c-Jun on the promoter of MMP9 . PANC-1 cells were transfected with empty siCtrl, siTNC, vector or TNC plasmid. PCR amplification from the total chromatin (bottom) was used as a positive control, anti-IgG (middle) served as a negative control, and anti-c-Jun (top) showed the interaction between the c-Jun and MMP9 promoter after the indicated treatment. (C) TNC transactivates the MMP9 promoter via c-Jun. The luciferase activity of the reporters in the indicated cells was assessed by a dual-luciferase reporter assay. The relative luciferase activity is the ratio of the luciferase activity in each of the tested cells to that in the control cells. Data represent the mean ± SD. (n = 3, * P
Figure Legend Snippet: C-Jun was required for TNC-induced MMP9 expression (A) The sequence and position of the c-Jun binding site in the MMP9 promoter are shown. (B) ChIP of c-Jun on the promoter of MMP9 . PANC-1 cells were transfected with empty siCtrl, siTNC, vector or TNC plasmid. PCR amplification from the total chromatin (bottom) was used as a positive control, anti-IgG (middle) served as a negative control, and anti-c-Jun (top) showed the interaction between the c-Jun and MMP9 promoter after the indicated treatment. (C) TNC transactivates the MMP9 promoter via c-Jun. The luciferase activity of the reporters in the indicated cells was assessed by a dual-luciferase reporter assay. The relative luciferase activity is the ratio of the luciferase activity in each of the tested cells to that in the control cells. Data represent the mean ± SD. (n = 3, * P

Techniques Used: Expressing, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control, Luciferase, Activity Assay, Reporter Assay

14) Product Images from "Essential Role of the Homeodomain for Pituitary Homeobox 1 Activation of Mouse Gonadotropin-Releasing Hormone Receptor Gene Expression through Interactions with c-Jun and DNA"

Article Title: Essential Role of the Homeodomain for Pituitary Homeobox 1 Activation of Mouse Gonadotropin-Releasing Hormone Receptor Gene Expression through Interactions with c-Jun and DNA

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.14.6127-6139.2004

Pitx-1 interacts directly with c-Jun. (A) EMSA was performed using GST/Pitx-1 and hc-Jun. −300/−259 was used as the probe. Each combination of GST/Pitx-1 (500 ng/lane), hc-Jun (0.1 fpu/lane), α-Pitx-1 antibody, and/or α-c-Jun antibody was incubated with probe. GST (500 ng/lane) was used as a negative control. *, new protein-DNA complex in the presence of both Pitx-1 and c-Jun. (B) GST pull-down analysis using hc-Jun (0.1 fpu/lane) and GST/Pitx-1 (1 μg/lane) was performed as described in Materials and Methods. GST (1 μg/lane) was used as a negative control. For the 10% input lane, 0.01 fpu of hc-Jun was loaded. (C) Luc reporter (pT109) containing 5xUAS was cotransfected into CV-1 cells with either Pitx-1 or empty expression vector and the indicated GAL4 DBD fusion constructs. Data are plotted as induction by Pitx-1 over the empty expression vector control response for each GAL4 DBD fusion construct. Data are presented as the mean ± SEM of three independent experiments. *, P
Figure Legend Snippet: Pitx-1 interacts directly with c-Jun. (A) EMSA was performed using GST/Pitx-1 and hc-Jun. −300/−259 was used as the probe. Each combination of GST/Pitx-1 (500 ng/lane), hc-Jun (0.1 fpu/lane), α-Pitx-1 antibody, and/or α-c-Jun antibody was incubated with probe. GST (500 ng/lane) was used as a negative control. *, new protein-DNA complex in the presence of both Pitx-1 and c-Jun. (B) GST pull-down analysis using hc-Jun (0.1 fpu/lane) and GST/Pitx-1 (1 μg/lane) was performed as described in Materials and Methods. GST (1 μg/lane) was used as a negative control. For the 10% input lane, 0.01 fpu of hc-Jun was loaded. (C) Luc reporter (pT109) containing 5xUAS was cotransfected into CV-1 cells with either Pitx-1 or empty expression vector and the indicated GAL4 DBD fusion constructs. Data are plotted as induction by Pitx-1 over the empty expression vector control response for each GAL4 DBD fusion construct. Data are presented as the mean ± SEM of three independent experiments. *, P

Techniques Used: Incubation, Negative Control, Expressing, Plasmid Preparation, Construct

Pitx-1 binds to the mGnRHR gene promoter in vivo but does not have distinct binding sites. (A) ChIP analysis was performed using LβT2 gonadotrope cells. After cross-linking, IP was performed by incubating with preimmune IgG, α-c-Jun, or α-Pitx-1 antibody. Input sample was prepared without IP and was further diluted to 1:100 for PCR. IgG, preimmune IgG; φ, water control for PCR. (B) Uracil (left panel) and methylation (right panel) interference analyses were performed using GST/Pitx-1 as described in Materials and Methods. hc-Jun was used as a positive control, binding to the AP-1 site. Locations of designated cis -elements are marked with vertical bars. F, free probe; P, GST/Pitx-1; J, c-Jun; *, c-Jun footprint.
Figure Legend Snippet: Pitx-1 binds to the mGnRHR gene promoter in vivo but does not have distinct binding sites. (A) ChIP analysis was performed using LβT2 gonadotrope cells. After cross-linking, IP was performed by incubating with preimmune IgG, α-c-Jun, or α-Pitx-1 antibody. Input sample was prepared without IP and was further diluted to 1:100 for PCR. IgG, preimmune IgG; φ, water control for PCR. (B) Uracil (left panel) and methylation (right panel) interference analyses were performed using GST/Pitx-1 as described in Materials and Methods. hc-Jun was used as a positive control, binding to the AP-1 site. Locations of designated cis -elements are marked with vertical bars. F, free probe; P, GST/Pitx-1; J, c-Jun; *, c-Jun footprint.

Techniques Used: In Vivo, Binding Assay, Chromatin Immunoprecipitation, Cross-linking Immunoprecipitation, Polymerase Chain Reaction, Methylation, Positive Control

15) Product Images from "Growth Factor Stimulation Induces Cell Survival by c-Jun?ATF2-dependent Activation of Bcl-XL *"

Article Title: Growth Factor Stimulation Induces Cell Survival by c-Jun?ATF2-dependent Activation of Bcl-XL *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.087221

ATF2 binds the Bcl-X proximal promoter and is necessary for the full activation of transcription. A , chromatin was isolated from HUVEC starved for 16 h and subsequently VEGF-treated for 2 h. ChIP assays were performed with HUVEC chromatin and specific antibodies recognizing the indicated c-Jun heterodimerization partners, control IgG, and primers M554 and M555. ATF2 bound the Bcl-X L promoter region, and its binding was increased after VEGF stimulation. B , the same experiment was performed with chromatin obtained from EGF treated fibroblasts wild type ( Jun ( +/+ ) ) and knock-out for c-Jun ( Jun ( −/− ) ). The ATF2 binding on the Bcl-X proximal promoter was c-Jun-dependent. C , HUVEC were transfected with two independent ATF2 shRNA vectors ( shATF2 C13 and shATF2 C14 ) and for GFP as control ( shGFP ). 30 h later, the cells were starved for 16 h and subsequently induced with VEGF. Whole cell extracts were immunoblotted with specific antibodies for ATF2, Bcl-X L , and SP1 as loading control. D , HUVEC were cotransfected with luciferase reporter construct Bcl-X promoter region and shRNA expressing vectors targeting ATF2 and GFP as control. Knockdown of ATF2 repressed Bcl-X proximal promoter activity. E , HUVEC were transfected with luciferase reporter constructs carrying the wild type or mutated Bcl-X proximal promoter and subsequently used in ChIP assays with specific anti-c-ATF2 antibody, control IgG, and primers M537 and M273. F , HUVEC were induced at the indicated time points with VEGF, and whole cell extract was immunoprecipitated ( IP ) with specific anti-ATF2 antibody and immunoblotted ( IB ) with anti-ATF2 and anti-Thr(P) 69/71 ATF2 antibodies.
Figure Legend Snippet: ATF2 binds the Bcl-X proximal promoter and is necessary for the full activation of transcription. A , chromatin was isolated from HUVEC starved for 16 h and subsequently VEGF-treated for 2 h. ChIP assays were performed with HUVEC chromatin and specific antibodies recognizing the indicated c-Jun heterodimerization partners, control IgG, and primers M554 and M555. ATF2 bound the Bcl-X L promoter region, and its binding was increased after VEGF stimulation. B , the same experiment was performed with chromatin obtained from EGF treated fibroblasts wild type ( Jun ( +/+ ) ) and knock-out for c-Jun ( Jun ( −/− ) ). The ATF2 binding on the Bcl-X proximal promoter was c-Jun-dependent. C , HUVEC were transfected with two independent ATF2 shRNA vectors ( shATF2 C13 and shATF2 C14 ) and for GFP as control ( shGFP ). 30 h later, the cells were starved for 16 h and subsequently induced with VEGF. Whole cell extracts were immunoblotted with specific antibodies for ATF2, Bcl-X L , and SP1 as loading control. D , HUVEC were cotransfected with luciferase reporter construct Bcl-X promoter region and shRNA expressing vectors targeting ATF2 and GFP as control. Knockdown of ATF2 repressed Bcl-X proximal promoter activity. E , HUVEC were transfected with luciferase reporter constructs carrying the wild type or mutated Bcl-X proximal promoter and subsequently used in ChIP assays with specific anti-c-ATF2 antibody, control IgG, and primers M537 and M273. F , HUVEC were induced at the indicated time points with VEGF, and whole cell extract was immunoprecipitated ( IP ) with specific anti-ATF2 antibody and immunoblotted ( IB ) with anti-ATF2 and anti-Thr(P) 69/71 ATF2 antibodies.

Techniques Used: Activation Assay, Isolation, Chromatin Immunoprecipitation, Binding Assay, Knock-Out, Transfection, shRNA, Luciferase, Construct, Expressing, Activity Assay, Immunoprecipitation

16) Product Images from "The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis"

Article Title: The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005579

Role of C/EBPβ in the expression of LOC105374325 in podocytes. A, prediction of transcription factors binding to LOC105374325 promoter region; B, level of LOC105374325 in podocytes treated with ADR, C/EBPβ siRNA, c-JUN siRNA, ERα siRNA, p53 siRNA, STAT1 siRNA, STAT4 siRNA, and XBP-1 siRNA ( n = 3); C, level of C/EBPβ and p-C/EBPβ protein in podocytes treated with ADR and SB203580 ( n = 3); D, ChIP analysis of the binding between C/EBPβ and LOC105374325 promoter in podocytes treated with ADR ( n = 3); E, schematic of the constructed LOC105374325 promoter-luciferase reporter plasmids; F, normalized luciferase activity of reporter constructs in podocytes cotransfected with C/EBPβ plasmid ( n = 5); G, level of LOC105374325 in podocytes transfected with C/EBPβ plasmid ( n = 5); H and I, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes transfected with C/EBPβ plasmid ( n = 5); J, level of Bax and Bak protein in podocytes transfected with C/EBPβ plasmid ( n = 3); K, flow cytometric analysis of apoptotic cells in podocytes transfected with C/EBPβ plasmid ( n = 5); L and M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and C/EBPβ siRNA ( n = 5); N, level of Bax and Bak protein in podocytes treated with ADR and C/EBPβ siRNA ( n = 3); O, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and C/EBPβ siRNA ( n = 5). For statistical analysis, a two-tailed Student's t test was used for F , G, and K , and one-way ANOVA with Tukey's post hoc test was used for B , H , I , L , M, and O . *, p
Figure Legend Snippet: Role of C/EBPβ in the expression of LOC105374325 in podocytes. A, prediction of transcription factors binding to LOC105374325 promoter region; B, level of LOC105374325 in podocytes treated with ADR, C/EBPβ siRNA, c-JUN siRNA, ERα siRNA, p53 siRNA, STAT1 siRNA, STAT4 siRNA, and XBP-1 siRNA ( n = 3); C, level of C/EBPβ and p-C/EBPβ protein in podocytes treated with ADR and SB203580 ( n = 3); D, ChIP analysis of the binding between C/EBPβ and LOC105374325 promoter in podocytes treated with ADR ( n = 3); E, schematic of the constructed LOC105374325 promoter-luciferase reporter plasmids; F, normalized luciferase activity of reporter constructs in podocytes cotransfected with C/EBPβ plasmid ( n = 5); G, level of LOC105374325 in podocytes transfected with C/EBPβ plasmid ( n = 5); H and I, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes transfected with C/EBPβ plasmid ( n = 5); J, level of Bax and Bak protein in podocytes transfected with C/EBPβ plasmid ( n = 3); K, flow cytometric analysis of apoptotic cells in podocytes transfected with C/EBPβ plasmid ( n = 5); L and M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and C/EBPβ siRNA ( n = 5); N, level of Bax and Bak protein in podocytes treated with ADR and C/EBPβ siRNA ( n = 3); O, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and C/EBPβ siRNA ( n = 5). For statistical analysis, a two-tailed Student's t test was used for F , G, and K , and one-way ANOVA with Tukey's post hoc test was used for B , H , I , L , M, and O . *, p

Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Two Tailed Test

17) Product Images from "Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7"

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144175

Curcumin prevented the binding of AP-1 to the functionally important AP1-1 site carried in the S100A7 promoter region. Biotin-labelled DNA fragments compatible with the sequence as -587 to -568 nucleotides of S100A7 promoter carrying either wild-type or mutated AP1-1 binding site were used to attach AP-1 complexes contained in the nuclear extracts prepared from HaCaT cells pre-incubated with PMA (lane(s) 2 and 6), curcumin (lane(s) 3 and 7) or combinations (lane(s) 4 and 8). The DNA-AP-1 complexes were immunoprecipitated by anti-c-Fos antibody than further processed for western blotting analysis since AP1-1 site acts as the predominant functional site for c-Jun/c-Fos to activate S100A7 promoter. ( A ) The presence of c-Fos at the wild-type AP1-1site was analyzed by western-blotting using anti-c-Fos antibody. Shown in lane(s) 1–4 are the 5% input of elute derived for each indicated condition. The amount of c-Fos recruited to the AP1-1 site is increased by the AP-1 stimulant PMA (comparing lane 6 to lane 1) but reduced by curcumin (compare lane 7 to lane 5). Moreover, the stimulatory effect derived from PMA treatment is also inhibited by curcumin (compare lane 8 to lane 6). ( B ) The presence of c-Fos at the mutated AP1-1site was also analyzed. Obviously, the recruitment of AP-1 is abolished by the mutagenesis performed on AP1-1 site (compare lane(s) 5–8 to lane 1~4). ( C ) The two PCR products from respective primer pairs covered the DNA fragment containing two AP-1 sites and indicated control region of endogenous S100A7 promoter were analyzed in the ChIP analysis. The different effect of curcumin were found using the respective antibody against different subunit of AP-1 complex, such as phosphorylated c-Fos and c-Jun. The results indicated more c-Jun-containing complexes will be recruited by curcumin for AP-1 sites occupancy in the endogenous S100A7 promoter. Results (A, B and C) are representative of two independent experiments.
Figure Legend Snippet: Curcumin prevented the binding of AP-1 to the functionally important AP1-1 site carried in the S100A7 promoter region. Biotin-labelled DNA fragments compatible with the sequence as -587 to -568 nucleotides of S100A7 promoter carrying either wild-type or mutated AP1-1 binding site were used to attach AP-1 complexes contained in the nuclear extracts prepared from HaCaT cells pre-incubated with PMA (lane(s) 2 and 6), curcumin (lane(s) 3 and 7) or combinations (lane(s) 4 and 8). The DNA-AP-1 complexes were immunoprecipitated by anti-c-Fos antibody than further processed for western blotting analysis since AP1-1 site acts as the predominant functional site for c-Jun/c-Fos to activate S100A7 promoter. ( A ) The presence of c-Fos at the wild-type AP1-1site was analyzed by western-blotting using anti-c-Fos antibody. Shown in lane(s) 1–4 are the 5% input of elute derived for each indicated condition. The amount of c-Fos recruited to the AP1-1 site is increased by the AP-1 stimulant PMA (comparing lane 6 to lane 1) but reduced by curcumin (compare lane 7 to lane 5). Moreover, the stimulatory effect derived from PMA treatment is also inhibited by curcumin (compare lane 8 to lane 6). ( B ) The presence of c-Fos at the mutated AP1-1site was also analyzed. Obviously, the recruitment of AP-1 is abolished by the mutagenesis performed on AP1-1 site (compare lane(s) 5–8 to lane 1~4). ( C ) The two PCR products from respective primer pairs covered the DNA fragment containing two AP-1 sites and indicated control region of endogenous S100A7 promoter were analyzed in the ChIP analysis. The different effect of curcumin were found using the respective antibody against different subunit of AP-1 complex, such as phosphorylated c-Fos and c-Jun. The results indicated more c-Jun-containing complexes will be recruited by curcumin for AP-1 sites occupancy in the endogenous S100A7 promoter. Results (A, B and C) are representative of two independent experiments.

Techniques Used: Binding Assay, Sequencing, Incubation, Immunoprecipitation, Western Blot, Functional Assay, Derivative Assay, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation

18) Product Images from "Activator Protein-1 Activation in Acute Lung Injury"

Article Title: Activator Protein-1 Activation in Acute Lung Injury

Journal: The American Journal of Pathology

doi:

A: Supershift analysis of AP-1 component in whole lung nuclear extract. DNA-binding reactions with nuclear extracts from whole lungs harvested 4 hours after IgG immune complex deposition were incubated with 32 P-labeled AP-1 oligonucleotide in the absence or presence of antibodies to the AP-1 proteins (c-fos, c-jun, jun-B, jun-D). B: Supershift analysis of AP-1 component in alveolar macrophages. DNA-binding reactions with nuclear extracts from alveolar macrophages harvested 0.5 hour after IgG immune complex deposition were incubated with 32 P-labeled AP-1 oligonucleotide in the absence or presence of antibodies to the AP-1 proteins (c-fos, c-jun, jun-B, jun-D). C: Western blot analysis of c-fos, c-jun, jun-B, and jun-D protein in nuclear extracts from alveolar macrophages (at 0 and 1 hour) and in lung nuclear extracts (at 0 and 4 hours) after onset of inflammation. The nuclear extracts (containing 25 μg of protein) were electrophoresed in a denaturing polyacrylamide gel and then transferred to a nitrocellulose membrane. Protein expression by Western blot analysis was evaluated by using the following antibodies: polyclonal rabbit anti-rat c-fos, polyclonal rabbit anti-rat c-jun, anti-rat jun-B, and polyclonal goat anti-rat jun-D. The graphs are representative of findings in two separate and independent experiments.
Figure Legend Snippet: A: Supershift analysis of AP-1 component in whole lung nuclear extract. DNA-binding reactions with nuclear extracts from whole lungs harvested 4 hours after IgG immune complex deposition were incubated with 32 P-labeled AP-1 oligonucleotide in the absence or presence of antibodies to the AP-1 proteins (c-fos, c-jun, jun-B, jun-D). B: Supershift analysis of AP-1 component in alveolar macrophages. DNA-binding reactions with nuclear extracts from alveolar macrophages harvested 0.5 hour after IgG immune complex deposition were incubated with 32 P-labeled AP-1 oligonucleotide in the absence or presence of antibodies to the AP-1 proteins (c-fos, c-jun, jun-B, jun-D). C: Western blot analysis of c-fos, c-jun, jun-B, and jun-D protein in nuclear extracts from alveolar macrophages (at 0 and 1 hour) and in lung nuclear extracts (at 0 and 4 hours) after onset of inflammation. The nuclear extracts (containing 25 μg of protein) were electrophoresed in a denaturing polyacrylamide gel and then transferred to a nitrocellulose membrane. Protein expression by Western blot analysis was evaluated by using the following antibodies: polyclonal rabbit anti-rat c-fos, polyclonal rabbit anti-rat c-jun, anti-rat jun-B, and polyclonal goat anti-rat jun-D. The graphs are representative of findings in two separate and independent experiments.

Techniques Used: Binding Assay, Incubation, Labeling, Western Blot, Expressing

19) Product Images from "Synergic induction of human periodontal ligament fibroblast cell death by nitric oxide and N-methyl-D-aspartic acid receptor antagonist"

Article Title: Synergic induction of human periodontal ligament fibroblast cell death by nitric oxide and N-methyl-D-aspartic acid receptor antagonist

Journal: Journal of Periodontal & Implant Science

doi: 10.5051/jpis.2011.41.1.17

Sodium nitroprusside (SNP) induced phosphorylation of mitogen-activated protein kinases in periodontal ligament fibroblasts (PDLFs). PDLFs were treated with SNP and (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801) for 16 hours. The cell lysates were performed to Western blot analysis using anti-c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SARK), anti-extracellular-signal-regulated kinase (ERK), anti-mitogen-activated protein kinases (p38), anti-phosphorylated JNK/SARK, anti-phosphorylated ERK or anti-phosphorylated p38 antibodies.
Figure Legend Snippet: Sodium nitroprusside (SNP) induced phosphorylation of mitogen-activated protein kinases in periodontal ligament fibroblasts (PDLFs). PDLFs were treated with SNP and (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801) for 16 hours. The cell lysates were performed to Western blot analysis using anti-c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SARK), anti-extracellular-signal-regulated kinase (ERK), anti-mitogen-activated protein kinases (p38), anti-phosphorylated JNK/SARK, anti-phosphorylated ERK or anti-phosphorylated p38 antibodies.

Techniques Used: Western Blot

20) Product Images from "Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants ▿ Mutants ▿ §"

Article Title: Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants ▿ Mutants ▿ §

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01330-08

(A) Coimmunoprecipitation assays were performed by immunoprecipitation (IP) with anti-c-Jun and blotting with anti-PU.1 and anti-c-Jun. To control for nuclear protein extract concentrations, levels of the nuclear protein TATA binding protein (TBP) were examined. (B) For ChIP assays, isolated lysates were immunoprecipitated with either anti-PU.1 or anti-c-Jun, followed by amplification of purified DNA fragments using primers specific for the c- Jun promoter or Mcsfr promoter or by antiacetylated H4, followed by amplification of purified DNA fragments using primers specific for the HPRT promoter.
Figure Legend Snippet: (A) Coimmunoprecipitation assays were performed by immunoprecipitation (IP) with anti-c-Jun and blotting with anti-PU.1 and anti-c-Jun. To control for nuclear protein extract concentrations, levels of the nuclear protein TATA binding protein (TBP) were examined. (B) For ChIP assays, isolated lysates were immunoprecipitated with either anti-PU.1 or anti-c-Jun, followed by amplification of purified DNA fragments using primers specific for the c- Jun promoter or Mcsfr promoter or by antiacetylated H4, followed by amplification of purified DNA fragments using primers specific for the HPRT promoter.

Techniques Used: Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Isolation, Amplification, Purification

21) Product Images from "Temporally distinct regulation of pathways contributing to cardiac proteostasis during the acute and recovery phases of sepsis"

Article Title: Temporally distinct regulation of pathways contributing to cardiac proteostasis during the acute and recovery phases of sepsis

Journal: Shock (Augusta, Ga.)

doi: 10.1097/SHK.0000000000001084

Schematic diagram of changes in proteostasis in the heart during the acute (top panel) and recovery (bottom panel) phases of sepsis produced by cecal ligation and puncture Thick solid lines and arrows indicate a stimulation of the respective pathway, whereas a thick dotted line indicates stimulation of an inhibitory pathway; thin lines indicate no detectable change in the activation state of the pathway. Abbreviations: 4E-BP1; eukaryotic initiation factor 4E binding protein-1; mTORC1, mammalian target of rapamycin complex 1; S6, ribosomal protein S6; S6K1, ribosome protein S6 kinase 1; UPP, ubiquitin proteasome pathway; eEF2, eukaryotic elongation factor; LC3B, light chain 3B; Atg, autophagy related protein; PARP, nuclear poly (ADP-ribose) polymerase; ATF4, activating transcription factor 4; JNK, c-Jun N-terminal kinase; NLRP3, NLR pyrin domain containing 3, TMS1, (aka ASC; adaptor protein containing a C-terminal caspase-recruitment domain; IL, interleukin; S, serine; T, threonine.
Figure Legend Snippet: Schematic diagram of changes in proteostasis in the heart during the acute (top panel) and recovery (bottom panel) phases of sepsis produced by cecal ligation and puncture Thick solid lines and arrows indicate a stimulation of the respective pathway, whereas a thick dotted line indicates stimulation of an inhibitory pathway; thin lines indicate no detectable change in the activation state of the pathway. Abbreviations: 4E-BP1; eukaryotic initiation factor 4E binding protein-1; mTORC1, mammalian target of rapamycin complex 1; S6, ribosomal protein S6; S6K1, ribosome protein S6 kinase 1; UPP, ubiquitin proteasome pathway; eEF2, eukaryotic elongation factor; LC3B, light chain 3B; Atg, autophagy related protein; PARP, nuclear poly (ADP-ribose) polymerase; ATF4, activating transcription factor 4; JNK, c-Jun N-terminal kinase; NLRP3, NLR pyrin domain containing 3, TMS1, (aka ASC; adaptor protein containing a C-terminal caspase-recruitment domain; IL, interleukin; S, serine; T, threonine.

Techniques Used: Produced, Ligation, Activation Assay, Binding Assay

22) Product Images from "Enhancer RNA and NFκB-dependent P300 regulation of ADAMDEC1"

Article Title: Enhancer RNA and NFκB-dependent P300 regulation of ADAMDEC1

Journal: Molecular immunology

doi: 10.1016/j.molimm.2018.09.019

ChIP-seq of the enhancer regions MonoMac6 cells were treated with 1μg/ml LPS for 90 minutes. ChIP assays were performed with antibodies H3K4me3, H3K27ac, c-JUN and P65. In resting cells, H3K4me3 was highest at Enhancer 1. LPS stimulation let to increased c-JUN at Enhancer 1, ADAM28 promoter, H3K27ac at the A DAMDEC1 promoter and Enhancer 2 as well as binding of p65 to the ADAM28 promoter and Enhancer 2. (n=4, error bars represent SD, * indicates p
Figure Legend Snippet: ChIP-seq of the enhancer regions MonoMac6 cells were treated with 1μg/ml LPS for 90 minutes. ChIP assays were performed with antibodies H3K4me3, H3K27ac, c-JUN and P65. In resting cells, H3K4me3 was highest at Enhancer 1. LPS stimulation let to increased c-JUN at Enhancer 1, ADAM28 promoter, H3K27ac at the A DAMDEC1 promoter and Enhancer 2 as well as binding of p65 to the ADAM28 promoter and Enhancer 2. (n=4, error bars represent SD, * indicates p

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

23) Product Images from "c-JUN Dimerization Protein 2 (JDP2) Is a Transcriptional Repressor of Follicle-stimulating Hormone β (FSHβ) and Is Required for Preventing Premature Reproductive Senescence in Female Mice *"

Article Title: c-JUN Dimerization Protein 2 (JDP2) Is a Transcriptional Repressor of Follicle-stimulating Hormone β (FSHβ) and Is Required for Preventing Premature Reproductive Senescence in Female Mice *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.771808

JDP2 interacts with NFY. A , GST pull-down assays demonstrate that NFY can interact directly with JDP2, ATF2, ATF3, and c-JUN, but not c-FOS. 35 S-Labeled proteins, indicated above each panel , were used in the binding assay with GST-NFY fusion protein or control GST, labeled above each panel . In the input panel , 10% of the in vitro transcribed and translated labeled proteins that were used in the binding reaction were run on the gel as a control for their expression and labeling. B , cells were transfected with histidine tag-JDP2 ( His ) or FLAG-tagged-c-FOS, and complexes were precipitated ( IP ) with antibodies to His tag or FLAG tag, run on the gel, and transferred, and membranes were probed with antibodies to c-JUN, c-FOS, and NFY-A to determine proteins that interact with JDP2. WB , Western blotting.
Figure Legend Snippet: JDP2 interacts with NFY. A , GST pull-down assays demonstrate that NFY can interact directly with JDP2, ATF2, ATF3, and c-JUN, but not c-FOS. 35 S-Labeled proteins, indicated above each panel , were used in the binding assay with GST-NFY fusion protein or control GST, labeled above each panel . In the input panel , 10% of the in vitro transcribed and translated labeled proteins that were used in the binding reaction were run on the gel as a control for their expression and labeling. B , cells were transfected with histidine tag-JDP2 ( His ) or FLAG-tagged-c-FOS, and complexes were precipitated ( IP ) with antibodies to His tag or FLAG tag, run on the gel, and transferred, and membranes were probed with antibodies to c-JUN, c-FOS, and NFY-A to determine proteins that interact with JDP2. WB , Western blotting.

Techniques Used: Labeling, Binding Assay, In Vitro, Expressing, Transfection, FLAG-tag, Western Blot

JDP2 repression of c-Jun and FSHβ gene expression. JDP2 is present in the gonadotrope at the basal level and inhibits c-JUN and FSHβ transcription, indicated with X . JDP2 binds c-JUN and FSHβ promoters at the CRE and TRE elements, respectively, and interacts with basal transcription factor NFY. JDP2 also interacts with ATF2 that binds the c-JUN promoter at the basal state alone and as a heterodimer with JDP2 ( top left ) and with c-JUN that is expressed at some basal level and binds the FSHβ promoter as a heterodimer with JDP2 ( bottom left ). GNRH treatment leads to phosphorylation and activation of ATF2 by p38 MAPK, which causes dissociation of JDP2 and induction of c-JUN transcription, indicated with an arrow ( top middle ). GNRH treatment also leads to induction of c-FOS via calcium calmodulin kinase II ( CKII ) activation, after which c-FOS displaces JDP2 as a c-JUN binding partner and activates FSHβ transcription ( bottom middle ). Dephosphorylation of ATF2 ( top right ) or degradation of c-FOS proteins due to their short half-life ( bottom right ) leads to recruitment of JDP2 and cessation of transcription until the next pulse of GNRH.
Figure Legend Snippet: JDP2 repression of c-Jun and FSHβ gene expression. JDP2 is present in the gonadotrope at the basal level and inhibits c-JUN and FSHβ transcription, indicated with X . JDP2 binds c-JUN and FSHβ promoters at the CRE and TRE elements, respectively, and interacts with basal transcription factor NFY. JDP2 also interacts with ATF2 that binds the c-JUN promoter at the basal state alone and as a heterodimer with JDP2 ( top left ) and with c-JUN that is expressed at some basal level and binds the FSHβ promoter as a heterodimer with JDP2 ( bottom left ). GNRH treatment leads to phosphorylation and activation of ATF2 by p38 MAPK, which causes dissociation of JDP2 and induction of c-JUN transcription, indicated with an arrow ( top middle ). GNRH treatment also leads to induction of c-FOS via calcium calmodulin kinase II ( CKII ) activation, after which c-FOS displaces JDP2 as a c-JUN binding partner and activates FSHβ transcription ( bottom middle ). Dephosphorylation of ATF2 ( top right ) or degradation of c-FOS proteins due to their short half-life ( bottom right ) leads to recruitment of JDP2 and cessation of transcription until the next pulse of GNRH.

Techniques Used: Expressing, Activation Assay, Binding Assay, De-Phosphorylation Assay

Threonine 148 is necessary for JDP2 repressor activity. c-JUN luciferase ( A ) or FSHβ luciferase ( B ) reporter were transfected into LβT2 cells with empty vector control, wild-type JDP2 ( JDP2 ), or JDP2 mutated at the threonine 148 to alanine ( T148A ) or to glutamic acid ( T148D ) or with JDP2 with a mutated DNA-binding domain ( DBDm ) and treated with vehicle or GNRH for 5 h. *, statistically significant difference ( p
Figure Legend Snippet: Threonine 148 is necessary for JDP2 repressor activity. c-JUN luciferase ( A ) or FSHβ luciferase ( B ) reporter were transfected into LβT2 cells with empty vector control, wild-type JDP2 ( JDP2 ), or JDP2 mutated at the threonine 148 to alanine ( T148A ) or to glutamic acid ( T148D ) or with JDP2 with a mutated DNA-binding domain ( DBDm ) and treated with vehicle or GNRH for 5 h. *, statistically significant difference ( p

Techniques Used: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Binding Assay

JDP2 binds FSHβ ( A and B ) and c-Jun ( C ) promoters. Nuclear extract from either LβT2 cells treated with GNRH for the times indicated above the lanes or COS-1 cells transfected with overexpression vectors for proteins indicated above the corresponding lanes was incubated with a 30-bp radiolabeled probe that encompasses the AP-1/TRE site in the FSHβ promoter ( A ), a 30-bp radiolabeled probe encompassing the AP-1/TRE site in the FSHβ promoter (TTGGTCA) or its mutation (TTGaaaA) compared with AP-1/TRE consensus (TGAGTCA) or its mutation (TGAaaaA) ( B ), or a 30-bp radiolabeled probe encompassing the CRE site in the c-JUN promoter ( C ). EMSA was performed three times, and representative gels subjected to autoradiography are shown.
Figure Legend Snippet: JDP2 binds FSHβ ( A and B ) and c-Jun ( C ) promoters. Nuclear extract from either LβT2 cells treated with GNRH for the times indicated above the lanes or COS-1 cells transfected with overexpression vectors for proteins indicated above the corresponding lanes was incubated with a 30-bp radiolabeled probe that encompasses the AP-1/TRE site in the FSHβ promoter ( A ), a 30-bp radiolabeled probe encompassing the AP-1/TRE site in the FSHβ promoter (TTGGTCA) or its mutation (TTGaaaA) compared with AP-1/TRE consensus (TGAGTCA) or its mutation (TGAaaaA) ( B ), or a 30-bp radiolabeled probe encompassing the CRE site in the c-JUN promoter ( C ). EMSA was performed three times, and representative gels subjected to autoradiography are shown.

Techniques Used: Transfection, Over Expression, Incubation, Mutagenesis, Autoradiography

24) Product Images from "Surgical Intervention for Symptomatic Benign Prostatic Hyperplasia is Correlated With Expression of the AP-1 Transcription Factor Network"

Article Title: Surgical Intervention for Symptomatic Benign Prostatic Hyperplasia is Correlated With Expression of the AP-1 Transcription Factor Network

Journal: The Prostate

doi: 10.1002/pros.22785

Expression of AP-1 transcription factors in Surgical versus Incidental BPH. Representative IHC images demonstrate increased epithelial and stromal cell expression of c-JUN ( A vs. B ), c-FOS ( C vs. D ). Western blot analysis displays increased activation
Figure Legend Snippet: Expression of AP-1 transcription factors in Surgical versus Incidental BPH. Representative IHC images demonstrate increased epithelial and stromal cell expression of c-JUN ( A vs. B ), c-FOS ( C vs. D ). Western blot analysis displays increased activation

Techniques Used: Expressing, Immunohistochemistry, Western Blot, Activation Assay

25) Product Images from "PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells"

Article Title: PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E06-09-0797

Interactions between unphosphorylated C terminus of c-Jun and Sp1 in cells. (A) Cells were transfected by lipofection with 2 μg of TAM-67, TAM-67-3A, and PP2B expression vectors, respectively. pcDNA3.1 was used as a vector to adjust for the same amount of plasmids in each experiment. Cells were incubated further for 36 h in the presence of 15 μM cyclosporin A (CsA). Nuclear extracts were prepared, immunoprecipitated with antibodies against c-Jun, and analyzed by Western blotting with anti-c-Jun and anti-Sp1 antibodies. (B) Densitometry analysis of the relative Sp1/TAM ratio was calculated. Values represent means ± SEM from three experiments. Statistical significance (*p
Figure Legend Snippet: Interactions between unphosphorylated C terminus of c-Jun and Sp1 in cells. (A) Cells were transfected by lipofection with 2 μg of TAM-67, TAM-67-3A, and PP2B expression vectors, respectively. pcDNA3.1 was used as a vector to adjust for the same amount of plasmids in each experiment. Cells were incubated further for 36 h in the presence of 15 μM cyclosporin A (CsA). Nuclear extracts were prepared, immunoprecipitated with antibodies against c-Jun, and analyzed by Western blotting with anti-c-Jun and anti-Sp1 antibodies. (B) Densitometry analysis of the relative Sp1/TAM ratio was calculated. Values represent means ± SEM from three experiments. Statistical significance (*p

Techniques Used: Transfection, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot

Interaction between Sp1 and unphosphorylated C terminus of c-Jun in vitro. (A) GST-TAM-67, GST-TAM-67-T231A, GST-TAM-67-S243/249A and GST-TAM-67-3A fusion proteins were expressed in E. coli and purified on glutathione-Sepharose. Each of the GST fusion proteins (1 μg) was in vitro phosphorylated by recombinant CKII and GSK3β, and repurified on glutathione-Sepharose. The proteins were separated by SDS-PAGE and visualized by autoradiography. The GST fusion proteins were detected by Coomassie blue. (B) Nuclear extracts were incubated with CKII-phosphorylated GST-fusion proteins and repurified on glutathione-Sepharose. The proteins were separated by SDS-PAGE and detected by anti-Sp1 and anti-c-Jun antibodies. (C) The phosphorylated and dephosphorylated forms of GST-c-Jun were incubated with PP2B and calmodulin purified from bovine brain in phosphatase buffer containing 1 mM Ca 2+ for 30 min at 37°C. Samples were repurified on GSH-Sepharose and then incubated with 500 μg of nuclear proteins for 30 min at 37°C. The proteins were purified on GSH-Sepharose again and separated by SDS-PAGE and detected by antibodies specific for PP2B, Sp1, and c-Jun. All panels show representative examples of three experiments with similar results.
Figure Legend Snippet: Interaction between Sp1 and unphosphorylated C terminus of c-Jun in vitro. (A) GST-TAM-67, GST-TAM-67-T231A, GST-TAM-67-S243/249A and GST-TAM-67-3A fusion proteins were expressed in E. coli and purified on glutathione-Sepharose. Each of the GST fusion proteins (1 μg) was in vitro phosphorylated by recombinant CKII and GSK3β, and repurified on glutathione-Sepharose. The proteins were separated by SDS-PAGE and visualized by autoradiography. The GST fusion proteins were detected by Coomassie blue. (B) Nuclear extracts were incubated with CKII-phosphorylated GST-fusion proteins and repurified on glutathione-Sepharose. The proteins were separated by SDS-PAGE and detected by anti-Sp1 and anti-c-Jun antibodies. (C) The phosphorylated and dephosphorylated forms of GST-c-Jun were incubated with PP2B and calmodulin purified from bovine brain in phosphatase buffer containing 1 mM Ca 2+ for 30 min at 37°C. Samples were repurified on GSH-Sepharose and then incubated with 500 μg of nuclear proteins for 30 min at 37°C. The proteins were purified on GSH-Sepharose again and separated by SDS-PAGE and detected by antibodies specific for PP2B, Sp1, and c-Jun. All panels show representative examples of three experiments with similar results.

Techniques Used: In Vitro, Purification, Recombinant, SDS Page, Autoradiography, Incubation

26) Product Images from "Inhibition by pentoxifylline of TNF-?-stimulated fractalkine production in vascular smooth muscle cells: evidence for mediation by NF-κB down-regulation"

Article Title: Inhibition by pentoxifylline of TNF-?-stimulated fractalkine production in vascular smooth muscle cells: evidence for mediation by NF-κB down-regulation

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0705088

Effects of pentoxifylline on TNF-α stimulated fractalkine mRNA and protein expression as well as TNF-α-activated phospho-p42/44 MAPK, phospho-PKC, phospho-c-Jun and I-κBα levels. VSMCs were incubated with TNF-α (5 ng ml −1 ) for 4 or 24 h, with or without pretreatment with PTX (pentoxifylline, 1–0.1 mg ml −1 ) for 45 min. (a) Representative Northern blots. FKN : fractalkine mRNA. (b) Representative Western blots. cFKN: cell-bound fractalkine, sFKN: soluble fractalkine. Lower panels show corresponding quantitative results of FKN/GAPDH mRNA and cFKN/β-actin ratios relative to that of control. Values are mean±s.e.mean of three experiments. * P
Figure Legend Snippet: Effects of pentoxifylline on TNF-α stimulated fractalkine mRNA and protein expression as well as TNF-α-activated phospho-p42/44 MAPK, phospho-PKC, phospho-c-Jun and I-κBα levels. VSMCs were incubated with TNF-α (5 ng ml −1 ) for 4 or 24 h, with or without pretreatment with PTX (pentoxifylline, 1–0.1 mg ml −1 ) for 45 min. (a) Representative Northern blots. FKN : fractalkine mRNA. (b) Representative Western blots. cFKN: cell-bound fractalkine, sFKN: soluble fractalkine. Lower panels show corresponding quantitative results of FKN/GAPDH mRNA and cFKN/β-actin ratios relative to that of control. Values are mean±s.e.mean of three experiments. * P

Techniques Used: Expressing, Incubation, Northern Blot, Western Blot

27) Product Images from "Binding Site Specificity and Factor Redundancy in Activator Protein-1-driven Human Papillomavirus Chromatin-dependent Transcription *"

Article Title: Binding Site Specificity and Factor Redundancy in Activator Protein-1-driven Human Papillomavirus Chromatin-dependent Transcription *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.290874

Recombinant full-length dimeric c-Jun-containing human AP-1 complexes are all active in binding the consensus TRE sequence found in the human cyclin D1 gene. A , shown is a schematic drawing of human c-Jun and Fos family proteins tagged at the N terminus
Figure Legend Snippet: Recombinant full-length dimeric c-Jun-containing human AP-1 complexes are all active in binding the consensus TRE sequence found in the human cyclin D1 gene. A , shown is a schematic drawing of human c-Jun and Fos family proteins tagged at the N terminus

Techniques Used: Recombinant, Binding Assay, Sequencing

28) Product Images from "Autocrine growth and anchorage independence: two complementing Jun-controlled genetic programs of cellular transformation"

Article Title: Autocrine growth and anchorage independence: two complementing Jun-controlled genetic programs of cellular transformation

Journal: Genes & Development

doi:

 Expression of wild-type and of mutant human c-Jun proteins in infected CEF cells measured by Western blot analysis. Total cell extract (10 μg) was subjected to SDS-PAGE, blotted onto nitrocellulose and incubated with a polyclonal antibody raised against bacterially expressed mouse c-Jun. The two arrows indicate the positions of the endogenous avian c-Jun protein (c-cJun, 314 amino acids; calculated molecular mass of 34.4 kD) and the virally expressed human c-Jun proteins (h-c-Jun, 331 amino acids; calculated molecular mass of 35.7 kD), respectively.
Figure Legend Snippet:  Expression of wild-type and of mutant human c-Jun proteins in infected CEF cells measured by Western blot analysis. Total cell extract (10 μg) was subjected to SDS-PAGE, blotted onto nitrocellulose and incubated with a polyclonal antibody raised against bacterially expressed mouse c-Jun. The two arrows indicate the positions of the endogenous avian c-Jun protein (c-cJun, 314 amino acids; calculated molecular mass of 34.4 kD) and the virally expressed human c-Jun proteins (h-c-Jun, 331 amino acids; calculated molecular mass of 35.7 kD), respectively.

Techniques Used: Expressing, Mutagenesis, Infection, Western Blot, SDS Page, Incubation

29) Product Images from "Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation"

Article Title: Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118473

S1P induces ICAM-1 expression via EGFR and PDGFR. (A) HPAEpiCs were pretreated with AG1296 or AG1478 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with AG1296 (10 M) or AG1478 (10 μM) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were transfected with siRNA of scrambled, EGFR, or PDGFR, and then incubated with S1P (10 μM) for 16 h. The levels of EGFR, PDGFR, and ICAM-1 proteins were determined by Western blot. (D, E) Cells were pretreated without or with AG1478, AG1296, or PP1 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-EGFR or phospho-PDGFR were determined by Western blot. (F) Cells were treated with S1P for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-c-Src, anti-EGFR, or anti-PDGFR antibody. The immunoprecipitates were analyzed by Western blot using an anti-PDGFR, anti-EGFR, or anti-c-Src antibody. Data are expressed as mean (C, D, E) or mean±S.E.M (A, B) of three independent experiments. * P
Figure Legend Snippet: S1P induces ICAM-1 expression via EGFR and PDGFR. (A) HPAEpiCs were pretreated with AG1296 or AG1478 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with AG1296 (10 M) or AG1478 (10 μM) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were transfected with siRNA of scrambled, EGFR, or PDGFR, and then incubated with S1P (10 μM) for 16 h. The levels of EGFR, PDGFR, and ICAM-1 proteins were determined by Western blot. (D, E) Cells were pretreated without or with AG1478, AG1296, or PP1 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-EGFR or phospho-PDGFR were determined by Western blot. (F) Cells were treated with S1P for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-c-Src, anti-EGFR, or anti-PDGFR antibody. The immunoprecipitates were analyzed by Western blot using an anti-PDGFR, anti-EGFR, or anti-c-Src antibody. Data are expressed as mean (C, D, E) or mean±S.E.M (A, B) of three independent experiments. * P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection, Immunoprecipitation

30) Product Images from "Schnurri-3 (KRC) Interacts with c-Jun to Regulate the IL-2 Gene in T Cells"

Article Title: Schnurri-3 (KRC) Interacts with c-Jun to Regulate the IL-2 Gene in T Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20030421

KRC physically interacts with c-Jun and acts as a transcriptional coactivator. (A) 293T cells were transfected with c-Jun and myc-KRCtr. 48 h later, lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were probed by Western blotting with anti–c-Jun antibody. (B) 293T cells were cotransfected with c-Jun and full length His-KRC (left). 48 h later, lysates were immunoprecipitated with anti-His antibody (DE8 Omniprobe), and precipitates were probed by Western blotting with anti–c-Jun antibody. In vitro–translated and S35-labeled c-Jun and His-KRCtr were mixed and immunoprecipitated with anti-His antibody (right). Recovered c-Jun protein was visualized by autoradiography. (C) Jurkat or EL4 T cells were stimulated with PMA plus ionomycin for 45 min. Lysates were immunoprecipitated with anti–c-Jun antibody, and immunoprecipitates were probed with specific anti-KRC rabbit antisera. (D) 293T cells were transfected with AP-1 luciferase along with c-Jun, c-Fos, and KRC (top). 24 h later, luciferase activity was determined as aforementioned. 293T cells were transfected with GAL4 luciferase along with GAL4, GAL4–c-Jun 1-224, or GAL4–c-Fos 208-313 (bottom). 24 h later, luciferase activity was determined as aforementioned.
Figure Legend Snippet: KRC physically interacts with c-Jun and acts as a transcriptional coactivator. (A) 293T cells were transfected with c-Jun and myc-KRCtr. 48 h later, lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were probed by Western blotting with anti–c-Jun antibody. (B) 293T cells were cotransfected with c-Jun and full length His-KRC (left). 48 h later, lysates were immunoprecipitated with anti-His antibody (DE8 Omniprobe), and precipitates were probed by Western blotting with anti–c-Jun antibody. In vitro–translated and S35-labeled c-Jun and His-KRCtr were mixed and immunoprecipitated with anti-His antibody (right). Recovered c-Jun protein was visualized by autoradiography. (C) Jurkat or EL4 T cells were stimulated with PMA plus ionomycin for 45 min. Lysates were immunoprecipitated with anti–c-Jun antibody, and immunoprecipitates were probed with specific anti-KRC rabbit antisera. (D) 293T cells were transfected with AP-1 luciferase along with c-Jun, c-Fos, and KRC (top). 24 h later, luciferase activity was determined as aforementioned. 293T cells were transfected with GAL4 luciferase along with GAL4, GAL4–c-Jun 1-224, or GAL4–c-Fos 208-313 (bottom). 24 h later, luciferase activity was determined as aforementioned.

Techniques Used: Transfection, Immunoprecipitation, Western Blot, In Vitro, Labeling, Autoradiography, Luciferase, Activity Assay

31) Product Images from "Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways"

Article Title: Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-015-0146-y

The MPP + -induced pro-apoptotic responses in CATH.a cells were attenuated by LSA. CATH.a cells were cultured with 300 μM MPP + for various time-periods ( a ) or pretreated with 100 μM LSA for 30 min, and then were cultured with 300 μM MPP + for 8 h ( b ). After treatments, the indicated proteins were detected by western blotting and the representative images are shown. Lower part of panel ( a ) and ( b ) are the relative protein level of GRP-78, caspase 7 (Casp7), caspase 9 (Casp9), cleaved caspase 3 (c-Casp3) and cleaved PARP (c-PARP). The protein level has been normalized with GAPDH. Results are means ± S.D. from three independent experiments. Significant differences between the cells treated with vehicle and MPP + were indicated by *, P
Figure Legend Snippet: The MPP + -induced pro-apoptotic responses in CATH.a cells were attenuated by LSA. CATH.a cells were cultured with 300 μM MPP + for various time-periods ( a ) or pretreated with 100 μM LSA for 30 min, and then were cultured with 300 μM MPP + for 8 h ( b ). After treatments, the indicated proteins were detected by western blotting and the representative images are shown. Lower part of panel ( a ) and ( b ) are the relative protein level of GRP-78, caspase 7 (Casp7), caspase 9 (Casp9), cleaved caspase 3 (c-Casp3) and cleaved PARP (c-PARP). The protein level has been normalized with GAPDH. Results are means ± S.D. from three independent experiments. Significant differences between the cells treated with vehicle and MPP + were indicated by *, P

Techniques Used: Cell Culture, Western Blot

32) Product Images from "Possible Involvement of Hepatitis B Virus Infection of Hepatocytes in the Attenuation of Apoptosis in Hepatic Stellate Cells"

Article Title: Possible Involvement of Hepatitis B Virus Infection of Hepatocytes in the Attenuation of Apoptosis in Hepatic Stellate Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146314

c-Jun is important for apoptotic hepatic stellate cell death induced by MG132. (A) Apoptotic cell deaths were lower in LX-2 cells transfected with siRNAs against c-Jun (si-c-Jun1 and si-c-Jun2) compared with LX-2 cells transfected with siRNA-control (si-C) after incubation with conditioned media from HepG2 in the presence of MG132 (lower panel). Western blot analysis of c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2 after 24 hours of MG132 treatment (upper panel). (B) Overexpression of c-Jun by the transfection of pMEKK into LX-2 cells enhanced apoptosis in LX-2 cells treated with conditioned media from HepG2.2.15 in the presence of MG132 (lower panel). Western blot analyses of c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2.2.15 after 24 hours of MG132 treatment (upper panel). Apoptosis was quantified using the APOPercentage Apoptosis Assay. Data are expressed as mean ± standard deviations of triplicate determinations. (C) Conditioned media from HepG2.2.15 (2.2.15-CM) protects hepatic stellate cells from MG132-induced apoptosis and DNA damage. Confocal microscopic findings with a high-power view (x200) of the expression of phosphorylated histone H2AX (γ-H2AX) (red), a DNA damage marker, and Annexin V (green), an apoptosis marker, in LX-2 cells treated with conditioned media from HepG2.2.15 (upper panel) or HepG2 (lower panel) in the presence of MG132.
Figure Legend Snippet: c-Jun is important for apoptotic hepatic stellate cell death induced by MG132. (A) Apoptotic cell deaths were lower in LX-2 cells transfected with siRNAs against c-Jun (si-c-Jun1 and si-c-Jun2) compared with LX-2 cells transfected with siRNA-control (si-C) after incubation with conditioned media from HepG2 in the presence of MG132 (lower panel). Western blot analysis of c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2 after 24 hours of MG132 treatment (upper panel). (B) Overexpression of c-Jun by the transfection of pMEKK into LX-2 cells enhanced apoptosis in LX-2 cells treated with conditioned media from HepG2.2.15 in the presence of MG132 (lower panel). Western blot analyses of c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2.2.15 after 24 hours of MG132 treatment (upper panel). Apoptosis was quantified using the APOPercentage Apoptosis Assay. Data are expressed as mean ± standard deviations of triplicate determinations. (C) Conditioned media from HepG2.2.15 (2.2.15-CM) protects hepatic stellate cells from MG132-induced apoptosis and DNA damage. Confocal microscopic findings with a high-power view (x200) of the expression of phosphorylated histone H2AX (γ-H2AX) (red), a DNA damage marker, and Annexin V (green), an apoptosis marker, in LX-2 cells treated with conditioned media from HepG2.2.15 (upper panel) or HepG2 (lower panel) in the presence of MG132.

Techniques Used: Transfection, Incubation, Western Blot, Expressing, Over Expression, Apoptosis Assay, Marker

33) Product Images from "TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation"

Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072783

TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P
Figure Legend Snippet: TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P

Techniques Used: Transfection, Incubation, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

34) Product Images from "Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation"

Article Title: Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.23-12-05187.2003

ATF3 expression rescues PC12 cells from ΔMEKK1-induced cell death and promotes neurite outgrowth. A , Expression of adenovirus-induced protein in infected PC12 cells. ATF3 and ΔMEKK1 were detected by Western blotting analysis using anti-c-myc antibody. Note that similar levels of expression are achieved in both single and double infections. B , Viability of PC12 cells 48 hr after adenovirus infection compared with uninfected PC12 cells. Viability of cells without adenovirus infection was defined as 100%. Each column represents the average of values from four different experiments. C , Morphology of adenovirus-infected PC12 cells. Forty-eight hours after adenovirus infection, the cells were fixed, and infected cells were detected with anti-β-galactosidase antibody (LacZ) or anti-c-myc antibody (ATF3 and ΔMEKK1) (green). Phalloidin conjugated with Alexa594 was used to visualize the cell shape. Scale bar, 20 μm. D , Quantification of neurite outgrowth. The percentage of the cells with neurite exceeding twice the cell body diameter length among the infected cells is shown. E – H , Immunoblot analysis of PC12 cells after adenovirus infection. In PC12 cells, phosphorylation of JNK was increased after ΔMEKK1 infection, whereas phosphorylation of ERKs and p38 was not changed. Phosphorylation of Akt was increased when ad-ATF3 and ad-ΔMEKK1 were coinfected. The expression levels of ERK and Akt are not altered by the viral infections.
Figure Legend Snippet: ATF3 expression rescues PC12 cells from ΔMEKK1-induced cell death and promotes neurite outgrowth. A , Expression of adenovirus-induced protein in infected PC12 cells. ATF3 and ΔMEKK1 were detected by Western blotting analysis using anti-c-myc antibody. Note that similar levels of expression are achieved in both single and double infections. B , Viability of PC12 cells 48 hr after adenovirus infection compared with uninfected PC12 cells. Viability of cells without adenovirus infection was defined as 100%. Each column represents the average of values from four different experiments. C , Morphology of adenovirus-infected PC12 cells. Forty-eight hours after adenovirus infection, the cells were fixed, and infected cells were detected with anti-β-galactosidase antibody (LacZ) or anti-c-myc antibody (ATF3 and ΔMEKK1) (green). Phalloidin conjugated with Alexa594 was used to visualize the cell shape. Scale bar, 20 μm. D , Quantification of neurite outgrowth. The percentage of the cells with neurite exceeding twice the cell body diameter length among the infected cells is shown. E – H , Immunoblot analysis of PC12 cells after adenovirus infection. In PC12 cells, phosphorylation of JNK was increased after ΔMEKK1 infection, whereas phosphorylation of ERKs and p38 was not changed. Phosphorylation of Akt was increased when ad-ATF3 and ad-ΔMEKK1 were coinfected. The expression levels of ERK and Akt are not altered by the viral infections.

Techniques Used: Expressing, Infection, Western Blot

ATF3 expression rescues SCG neurons from NGF deprivation-induced neuronal death and elongates neurite. A, a , Viability of adenovirus-infected SCG neurons 48 hr after NGF withdrawal. Viability of SCG neurons cultured with NGF medium was defined as 100%. Each column represents the average of values from four different experiments. * p = 0.01 (Student's t test). b , NGF was withdrawn 24 hr after adenovirus infection, and another 24 hr later, nuclear morphology was visualized with Hoechst 33342. The ratio of cells having pyknotic nuclei among adenovirus-expressing cells visualized by anti-c-myc or anti-β-galactosidase antibody was measured. * p = 0.01 (Student's t test). B—G , Morphology of adenovirus-infected SCGs ( B – D , ad-ATF3; E – G , ad-LacZ). After being cultured without growth factors for 5 d, the cells were fixed and stained with anti-neurofilament antibody (red). Anti-c-Myc antibody and anti-β-galactosidase antibody were used to detect adenovirus-infected neurons (green). Scalebar, 100 μm. H , Neurite length of infected cells was measured from the organ cultured edge of SCG. I – K , Immunoblot analysis for the expressions of pJNK, pERK, and pAkt in SCG neurons after the adenovirus infection. Twenty-four hours after adenovirus infection, SCG neurons were refed with +NGF or -NGF medium and incubated for 1 more day. When NGF was depleted, JNK activation (increase of pJNK level) was seen. ATF3 expression under the condition of -NGF induced Akt activation (pAkt) but not ERK ( J, K ).
Figure Legend Snippet: ATF3 expression rescues SCG neurons from NGF deprivation-induced neuronal death and elongates neurite. A, a , Viability of adenovirus-infected SCG neurons 48 hr after NGF withdrawal. Viability of SCG neurons cultured with NGF medium was defined as 100%. Each column represents the average of values from four different experiments. * p = 0.01 (Student's t test). b , NGF was withdrawn 24 hr after adenovirus infection, and another 24 hr later, nuclear morphology was visualized with Hoechst 33342. The ratio of cells having pyknotic nuclei among adenovirus-expressing cells visualized by anti-c-myc or anti-β-galactosidase antibody was measured. * p = 0.01 (Student's t test). B—G , Morphology of adenovirus-infected SCGs ( B – D , ad-ATF3; E – G , ad-LacZ). After being cultured without growth factors for 5 d, the cells were fixed and stained with anti-neurofilament antibody (red). Anti-c-Myc antibody and anti-β-galactosidase antibody were used to detect adenovirus-infected neurons (green). Scalebar, 100 μm. H , Neurite length of infected cells was measured from the organ cultured edge of SCG. I – K , Immunoblot analysis for the expressions of pJNK, pERK, and pAkt in SCG neurons after the adenovirus infection. Twenty-four hours after adenovirus infection, SCG neurons were refed with +NGF or -NGF medium and incubated for 1 more day. When NGF was depleted, JNK activation (increase of pJNK level) was seen. ATF3 expression under the condition of -NGF induced Akt activation (pAkt) but not ERK ( J, K ).

Techniques Used: Expressing, Infection, Cell Culture, Staining, Incubation, Activation Assay

ATF3 binds to c-Jun and activates Hsp27 promoter and the transcription. A , Immunoprecipitation analysis shows that ATF3 binds to c-Jun in PC12 cells. PC12 cells were infected with ad-ATF3 (c-Myc tagged) and adenovirus overexpressing c-Jun (ad-c-Jun, HA tagged). Forty-eight hours after infection, cells were lysed and immunoprecipitated using anti-HA antibody or anti-c-Myc antibody. B – D , Characterization of C-terminal truncated form of c-Jun (c-Jun223NLS), as a c-Jun dominant-negative form. B , C-terminal truncated form of c-Jun (c-Jun223NLS) suppresses the phosphorylation of endogenous c-Jun. Phosphorylation of endogenous c-Jun is increased after adΔMEKK1 infection (arrow a ), but its increase is suppressed in additional infection of adc-Jun223NLS. Arrow b indicates phosphorylation of c-Jun223NLS (∼25 kDa). HA-tagged c-Jun223NLS was used in this analysis and detected by anti-HA antibody. C, D , A dominant-negative effect of c-Jun223NLS on promoters having conservative AP-1 ( C ) and CRE ( D ) motifs. The adenovirus-infected PC12 cells (12 hr after infection) were transfected with luciferase (Luc) reporter vector, which contains conservative AP-1 or CRE motif. Twenty-four hours after transfection, cell extract was assayed for luciferase activity. The averages of activities from three experiments are shown. The values obtained from ad-LacZ-infected cells were defined as 100%. E , C-terminal truncated form of c-Jun (c-Jun223NLS) diminishes ΔMEKK1- and ATF3-induced activity of Hsp27 promoter. Hsp27-Luc was transfected into PC12 cells together with plasmids expressing ATF3, ΔMEKK1, and c-Jun223NLS. The values obtained from noninfected cells were defined as 100%. F , RT-PCR analysis of Hsp27 mRNA expression in PC12 cells (48 hr after adenovirus infection) and SCG neurons (incubated with +NGF or -NGF medium for 1 d, 24 hr after adenovirus infection). Hsp27 mRNA upregulation by ATF3 was diminished when c-Jun223NLS was coexpressed.
Figure Legend Snippet: ATF3 binds to c-Jun and activates Hsp27 promoter and the transcription. A , Immunoprecipitation analysis shows that ATF3 binds to c-Jun in PC12 cells. PC12 cells were infected with ad-ATF3 (c-Myc tagged) and adenovirus overexpressing c-Jun (ad-c-Jun, HA tagged). Forty-eight hours after infection, cells were lysed and immunoprecipitated using anti-HA antibody or anti-c-Myc antibody. B – D , Characterization of C-terminal truncated form of c-Jun (c-Jun223NLS), as a c-Jun dominant-negative form. B , C-terminal truncated form of c-Jun (c-Jun223NLS) suppresses the phosphorylation of endogenous c-Jun. Phosphorylation of endogenous c-Jun is increased after adΔMEKK1 infection (arrow a ), but its increase is suppressed in additional infection of adc-Jun223NLS. Arrow b indicates phosphorylation of c-Jun223NLS (∼25 kDa). HA-tagged c-Jun223NLS was used in this analysis and detected by anti-HA antibody. C, D , A dominant-negative effect of c-Jun223NLS on promoters having conservative AP-1 ( C ) and CRE ( D ) motifs. The adenovirus-infected PC12 cells (12 hr after infection) were transfected with luciferase (Luc) reporter vector, which contains conservative AP-1 or CRE motif. Twenty-four hours after transfection, cell extract was assayed for luciferase activity. The averages of activities from three experiments are shown. The values obtained from ad-LacZ-infected cells were defined as 100%. E , C-terminal truncated form of c-Jun (c-Jun223NLS) diminishes ΔMEKK1- and ATF3-induced activity of Hsp27 promoter. Hsp27-Luc was transfected into PC12 cells together with plasmids expressing ATF3, ΔMEKK1, and c-Jun223NLS. The values obtained from noninfected cells were defined as 100%. F , RT-PCR analysis of Hsp27 mRNA expression in PC12 cells (48 hr after adenovirus infection) and SCG neurons (incubated with +NGF or -NGF medium for 1 d, 24 hr after adenovirus infection). Hsp27 mRNA upregulation by ATF3 was diminished when c-Jun223NLS was coexpressed.

Techniques Used: Immunoprecipitation, Infection, Dominant Negative Mutation, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

35) Product Images from "c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs"

Article Title: c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1323

In vivo binding of c-Jun/c-Fos to cellular promoters with meAP-1 sites in activated primary human B cells. After immunoprecipitation (ChIP) of chromatin from activated naïve B cells with an α-c-Jun antibody, the enrichment of the indicated promoter regions was assayed by quantitative PCR. Shown are the results obtained from three promoter regions indicated in red that contain conventional previously identified AP-1 sites ( 34 ) and three promoter regions indicated in black, which were identified in this study and are devoid of conventional AP-1 or meAP-1 sites. Both sets of promoters served as positive and negative controls, respectively. Ten promoters that were found to contain meAP-1 sites (and lack conventional AP-1 sites in proximity) were analyzed by ChIP and the results are shown in green. The PSMC1 promoter contains two separate regions with one meAP-1 site each depicted as PSMC1-1 and -2, which were analyzed separately. Details of all promoters investigated here are listed in Table 1 . Means and standard deviations of three independent experiments are shown.
Figure Legend Snippet: In vivo binding of c-Jun/c-Fos to cellular promoters with meAP-1 sites in activated primary human B cells. After immunoprecipitation (ChIP) of chromatin from activated naïve B cells with an α-c-Jun antibody, the enrichment of the indicated promoter regions was assayed by quantitative PCR. Shown are the results obtained from three promoter regions indicated in red that contain conventional previously identified AP-1 sites ( 34 ) and three promoter regions indicated in black, which were identified in this study and are devoid of conventional AP-1 or meAP-1 sites. Both sets of promoters served as positive and negative controls, respectively. Ten promoters that were found to contain meAP-1 sites (and lack conventional AP-1 sites in proximity) were analyzed by ChIP and the results are shown in green. The PSMC1 promoter contains two separate regions with one meAP-1 site each depicted as PSMC1-1 and -2, which were analyzed separately. Details of all promoters investigated here are listed in Table 1 . Means and standard deviations of three independent experiments are shown.

Techniques Used: In Vivo, Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

36) Product Images from "Sanguinarine protects against osteoarthritis by suppressing the expression of catabolic proteases"

Article Title: Sanguinarine protects against osteoarthritis by suppressing the expression of catabolic proteases

Journal: Oncotarget

doi: 10.18632/oncotarget.17036

Sanguinarine (SA) inhibits interleukin (IL)-1β-induced nuclear factor (NF)-κB and c-Jun N-terminal kinase (JNK) activation in chondrocytes (A) Chondrocytes were treated with or without 1.25 μm SA for 4 h and then 10 ng/mL IL-1β for indicated time. Phosphorylated (p)-p38, extracellular signal-regulated kinase (ERK), JNK, inhibitor of NF-Κb (IκB)-α, total p38, ERK, JNK, IκB-α, and β-actin were evaluated using western blot analysis. (B) Signal intensities of p-P38, ERK, JNK, and IκB-α were quantified and normalized to total ERK, P38, JNK, and IκB-α using ImageJ, n = 6, *p
Figure Legend Snippet: Sanguinarine (SA) inhibits interleukin (IL)-1β-induced nuclear factor (NF)-κB and c-Jun N-terminal kinase (JNK) activation in chondrocytes (A) Chondrocytes were treated with or without 1.25 μm SA for 4 h and then 10 ng/mL IL-1β for indicated time. Phosphorylated (p)-p38, extracellular signal-regulated kinase (ERK), JNK, inhibitor of NF-Κb (IκB)-α, total p38, ERK, JNK, IκB-α, and β-actin were evaluated using western blot analysis. (B) Signal intensities of p-P38, ERK, JNK, and IκB-α were quantified and normalized to total ERK, P38, JNK, and IκB-α using ImageJ, n = 6, *p

Techniques Used: Activation Assay, Western Blot

37) Product Images from "Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants ▿ Mutants ▿ §"

Article Title: Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants ▿ Mutants ▿ §

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01330-08

(A) Coimmunoprecipitation assays were performed by immunoprecipitation (IP) with anti-c-Jun and blotting with anti-PU.1 and anti-c-Jun. To control for nuclear protein extract concentrations, levels of the nuclear protein TATA binding protein (TBP) were examined. (B) For ChIP assays, isolated lysates were immunoprecipitated with either anti-PU.1 or anti-c-Jun, followed by amplification of purified DNA fragments using primers specific for the c- Jun promoter or Mcsfr promoter or by antiacetylated H4, followed by amplification of purified DNA fragments using primers specific for the HPRT promoter.
Figure Legend Snippet: (A) Coimmunoprecipitation assays were performed by immunoprecipitation (IP) with anti-c-Jun and blotting with anti-PU.1 and anti-c-Jun. To control for nuclear protein extract concentrations, levels of the nuclear protein TATA binding protein (TBP) were examined. (B) For ChIP assays, isolated lysates were immunoprecipitated with either anti-PU.1 or anti-c-Jun, followed by amplification of purified DNA fragments using primers specific for the c- Jun promoter or Mcsfr promoter or by antiacetylated H4, followed by amplification of purified DNA fragments using primers specific for the HPRT promoter.

Techniques Used: Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Isolation, Amplification, Purification

38) Product Images from "Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation"

Article Title: Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.23-12-05187.2003

ATF3 expression rescues PC12 cells from ΔMEKK1-induced cell death and promotes neurite outgrowth. A , Expression of adenovirus-induced protein in infected PC12 cells. ATF3 and ΔMEKK1 were detected by Western blotting analysis using anti-c-myc antibody. Note that similar levels of expression are achieved in both single and double infections. B , Viability of PC12 cells 48 hr after adenovirus infection compared with uninfected PC12 cells. Viability of cells without adenovirus infection was defined as 100%. Each column represents the average of values from four different experiments. C , Morphology of adenovirus-infected PC12 cells. Forty-eight hours after adenovirus infection, the cells were fixed, and infected cells were detected with anti-β-galactosidase antibody (LacZ) or anti-c-myc antibody (ATF3 and ΔMEKK1) (green). Phalloidin conjugated with Alexa594 was used to visualize the cell shape. Scale bar, 20 μm. D , Quantification of neurite outgrowth. The percentage of the cells with neurite exceeding twice the cell body diameter length among the infected cells is shown. E – H , Immunoblot analysis of PC12 cells after adenovirus infection. In PC12 cells, phosphorylation of JNK was increased after ΔMEKK1 infection, whereas phosphorylation of ERKs and p38 was not changed. Phosphorylation of Akt was increased when ad-ATF3 and ad-ΔMEKK1 were coinfected. The expression levels of ERK and Akt are not altered by the viral infections.
Figure Legend Snippet: ATF3 expression rescues PC12 cells from ΔMEKK1-induced cell death and promotes neurite outgrowth. A , Expression of adenovirus-induced protein in infected PC12 cells. ATF3 and ΔMEKK1 were detected by Western blotting analysis using anti-c-myc antibody. Note that similar levels of expression are achieved in both single and double infections. B , Viability of PC12 cells 48 hr after adenovirus infection compared with uninfected PC12 cells. Viability of cells without adenovirus infection was defined as 100%. Each column represents the average of values from four different experiments. C , Morphology of adenovirus-infected PC12 cells. Forty-eight hours after adenovirus infection, the cells were fixed, and infected cells were detected with anti-β-galactosidase antibody (LacZ) or anti-c-myc antibody (ATF3 and ΔMEKK1) (green). Phalloidin conjugated with Alexa594 was used to visualize the cell shape. Scale bar, 20 μm. D , Quantification of neurite outgrowth. The percentage of the cells with neurite exceeding twice the cell body diameter length among the infected cells is shown. E – H , Immunoblot analysis of PC12 cells after adenovirus infection. In PC12 cells, phosphorylation of JNK was increased after ΔMEKK1 infection, whereas phosphorylation of ERKs and p38 was not changed. Phosphorylation of Akt was increased when ad-ATF3 and ad-ΔMEKK1 were coinfected. The expression levels of ERK and Akt are not altered by the viral infections.

Techniques Used: Expressing, Infection, Western Blot

ATF3 expression rescues SCG neurons from NGF deprivation-induced neuronal death and elongates neurite. A, a , Viability of adenovirus-infected SCG neurons 48 hr after NGF withdrawal. Viability of SCG neurons cultured with NGF medium was defined as 100%. Each column represents the average of values from four different experiments. * p = 0.01 (Student's t test). b , NGF was withdrawn 24 hr after adenovirus infection, and another 24 hr later, nuclear morphology was visualized with Hoechst 33342. The ratio of cells having pyknotic nuclei among adenovirus-expressing cells visualized by anti-c-myc or anti-β-galactosidase antibody was measured. * p = 0.01 (Student's t test). B—G , Morphology of adenovirus-infected SCGs ( B – D , ad-ATF3; E – G , ad-LacZ). After being cultured without growth factors for 5 d, the cells were fixed and stained with anti-neurofilament antibody (red). Anti-c-Myc antibody and anti-β-galactosidase antibody were used to detect adenovirus-infected neurons (green). Scalebar, 100 μm. H , Neurite length of infected cells was measured from the organ cultured edge of SCG. I – K , Immunoblot analysis for the expressions of pJNK, pERK, and pAkt in SCG neurons after the adenovirus infection. Twenty-four hours after adenovirus infection, SCG neurons were refed with +NGF or -NGF medium and incubated for 1 more day. When NGF was depleted, JNK activation (increase of pJNK level) was seen. ATF3 expression under the condition of -NGF induced Akt activation (pAkt) but not ERK ( J, K ).
Figure Legend Snippet: ATF3 expression rescues SCG neurons from NGF deprivation-induced neuronal death and elongates neurite. A, a , Viability of adenovirus-infected SCG neurons 48 hr after NGF withdrawal. Viability of SCG neurons cultured with NGF medium was defined as 100%. Each column represents the average of values from four different experiments. * p = 0.01 (Student's t test). b , NGF was withdrawn 24 hr after adenovirus infection, and another 24 hr later, nuclear morphology was visualized with Hoechst 33342. The ratio of cells having pyknotic nuclei among adenovirus-expressing cells visualized by anti-c-myc or anti-β-galactosidase antibody was measured. * p = 0.01 (Student's t test). B—G , Morphology of adenovirus-infected SCGs ( B – D , ad-ATF3; E – G , ad-LacZ). After being cultured without growth factors for 5 d, the cells were fixed and stained with anti-neurofilament antibody (red). Anti-c-Myc antibody and anti-β-galactosidase antibody were used to detect adenovirus-infected neurons (green). Scalebar, 100 μm. H , Neurite length of infected cells was measured from the organ cultured edge of SCG. I – K , Immunoblot analysis for the expressions of pJNK, pERK, and pAkt in SCG neurons after the adenovirus infection. Twenty-four hours after adenovirus infection, SCG neurons were refed with +NGF or -NGF medium and incubated for 1 more day. When NGF was depleted, JNK activation (increase of pJNK level) was seen. ATF3 expression under the condition of -NGF induced Akt activation (pAkt) but not ERK ( J, K ).

Techniques Used: Expressing, Infection, Cell Culture, Staining, Incubation, Activation Assay

ATF3 binds to c-Jun and activates Hsp27 promoter and the transcription. A , Immunoprecipitation analysis shows that ATF3 binds to c-Jun in PC12 cells. PC12 cells were infected with ad-ATF3 (c-Myc tagged) and adenovirus overexpressing c-Jun (ad-c-Jun, HA tagged). Forty-eight hours after infection, cells were lysed and immunoprecipitated using anti-HA antibody or anti-c-Myc antibody. B – D , Characterization of C-terminal truncated form of c-Jun (c-Jun223NLS), as a c-Jun dominant-negative form. B , C-terminal truncated form of c-Jun (c-Jun223NLS) suppresses the phosphorylation of endogenous c-Jun. Phosphorylation of endogenous c-Jun is increased after adΔMEKK1 infection (arrow a ), but its increase is suppressed in additional infection of adc-Jun223NLS. Arrow b indicates phosphorylation of c-Jun223NLS (∼25 kDa). HA-tagged c-Jun223NLS was used in this analysis and detected by anti-HA antibody. C, D , A dominant-negative effect of c-Jun223NLS on promoters having conservative AP-1 ( C ) and CRE ( D ) motifs. The adenovirus-infected PC12 cells (12 hr after infection) were transfected with luciferase (Luc) reporter vector, which contains conservative AP-1 or CRE motif. Twenty-four hours after transfection, cell extract was assayed for luciferase activity. The averages of activities from three experiments are shown. The values obtained from ad-LacZ-infected cells were defined as 100%. E , C-terminal truncated form of c-Jun (c-Jun223NLS) diminishes ΔMEKK1- and ATF3-induced activity of Hsp27 promoter. Hsp27-Luc was transfected into PC12 cells together with plasmids expressing ATF3, ΔMEKK1, and c-Jun223NLS. The values obtained from noninfected cells were defined as 100%. F , RT-PCR analysis of Hsp27 mRNA expression in PC12 cells (48 hr after adenovirus infection) and SCG neurons (incubated with +NGF or -NGF medium for 1 d, 24 hr after adenovirus infection). Hsp27 mRNA upregulation by ATF3 was diminished when c-Jun223NLS was coexpressed.
Figure Legend Snippet: ATF3 binds to c-Jun and activates Hsp27 promoter and the transcription. A , Immunoprecipitation analysis shows that ATF3 binds to c-Jun in PC12 cells. PC12 cells were infected with ad-ATF3 (c-Myc tagged) and adenovirus overexpressing c-Jun (ad-c-Jun, HA tagged). Forty-eight hours after infection, cells were lysed and immunoprecipitated using anti-HA antibody or anti-c-Myc antibody. B – D , Characterization of C-terminal truncated form of c-Jun (c-Jun223NLS), as a c-Jun dominant-negative form. B , C-terminal truncated form of c-Jun (c-Jun223NLS) suppresses the phosphorylation of endogenous c-Jun. Phosphorylation of endogenous c-Jun is increased after adΔMEKK1 infection (arrow a ), but its increase is suppressed in additional infection of adc-Jun223NLS. Arrow b indicates phosphorylation of c-Jun223NLS (∼25 kDa). HA-tagged c-Jun223NLS was used in this analysis and detected by anti-HA antibody. C, D , A dominant-negative effect of c-Jun223NLS on promoters having conservative AP-1 ( C ) and CRE ( D ) motifs. The adenovirus-infected PC12 cells (12 hr after infection) were transfected with luciferase (Luc) reporter vector, which contains conservative AP-1 or CRE motif. Twenty-four hours after transfection, cell extract was assayed for luciferase activity. The averages of activities from three experiments are shown. The values obtained from ad-LacZ-infected cells were defined as 100%. E , C-terminal truncated form of c-Jun (c-Jun223NLS) diminishes ΔMEKK1- and ATF3-induced activity of Hsp27 promoter. Hsp27-Luc was transfected into PC12 cells together with plasmids expressing ATF3, ΔMEKK1, and c-Jun223NLS. The values obtained from noninfected cells were defined as 100%. F , RT-PCR analysis of Hsp27 mRNA expression in PC12 cells (48 hr after adenovirus infection) and SCG neurons (incubated with +NGF or -NGF medium for 1 d, 24 hr after adenovirus infection). Hsp27 mRNA upregulation by ATF3 was diminished when c-Jun223NLS was coexpressed.

Techniques Used: Immunoprecipitation, Infection, Dominant Negative Mutation, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

39) Product Images from "Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways"

Article Title: Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S71957

Effects of cordycepin on LPS-induced mitogen-activated protein kinase phosphorylation in RAW 264.7 macrophages. Notes: Cells were treated with 100 ng/mL LPS for the indicated times ( A ) or treated with different concentrations of cordycepin 1 hour before LPS treatment for 30 minutes ( B ). Total proteins were prepared and separated on 10% sodium dodecyl sulfate-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Abbreviations: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide.
Figure Legend Snippet: Effects of cordycepin on LPS-induced mitogen-activated protein kinase phosphorylation in RAW 264.7 macrophages. Notes: Cells were treated with 100 ng/mL LPS for the indicated times ( A ) or treated with different concentrations of cordycepin 1 hour before LPS treatment for 30 minutes ( B ). Total proteins were prepared and separated on 10% sodium dodecyl sulfate-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Abbreviations: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide.

Techniques Used: Western Blot

40) Product Images from "Adiponectin associates with markers of cartilage degradation in osteoarthritis and induces production of proinflammatory and catabolic factors through mitogen-activated protein kinase pathways"

Article Title: Adiponectin associates with markers of cartilage degradation in osteoarthritis and induces production of proinflammatory and catabolic factors through mitogen-activated protein kinase pathways

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3512

Adiponectin induces activation of mitogen-activated protein kinases in human primary chondrocytes . The effect of adiponectin (3 μg/ml) on mitogen-activated protein kinase (MAPK) phosphorylation in human primary chondrocytes obtained from patients with OA. The figure shows the results of a representative experiment which was repeated three times (that is, with cells from three donors) with similar results. MAPKs were determined by Western blot analysis at baseline and at 30 and 60 minutes after addition of adiponectin. Erk1/2 = extracellular signal-regulated kinase 1/2; JNK: c-Jun N-terminal kinase.
Figure Legend Snippet: Adiponectin induces activation of mitogen-activated protein kinases in human primary chondrocytes . The effect of adiponectin (3 μg/ml) on mitogen-activated protein kinase (MAPK) phosphorylation in human primary chondrocytes obtained from patients with OA. The figure shows the results of a representative experiment which was repeated three times (that is, with cells from three donors) with similar results. MAPKs were determined by Western blot analysis at baseline and at 30 and 60 minutes after addition of adiponectin. Erk1/2 = extracellular signal-regulated kinase 1/2; JNK: c-Jun N-terminal kinase.

Techniques Used: Activation Assay, Western Blot

41) Product Images from "A specific lysine in c-Jun is required for transcriptional repression by E1A and is acetylated by p300"

Article Title: A specific lysine in c-Jun is required for transcriptional repression by E1A and is acetylated by p300

Journal: The EMBO Journal

doi: 10.1093/emboj/20.21.6095

Fig. 3. Acetylation of c-Jun in vivo . c-Jun was immunoprecipitated with the H79 c-Jun antibody from Ad5HER cells. The cells were mock treated or treated with media supplemented with the acetyl-CoA precursor [ 14 C]pyruvate or [ 35 S]methionine for 1 h. The immunoprecipitations were analysed by a western blot with the Ab-1 anti-c-Jun antibody (1) and labelled immunoprecipitations by autoradiography of [ 35 S]methionine (2) and [ 14 C]pyruvate (3). As a control for the incorporation of 14 C-label in proteins in general, E1A was immunoprecipitated (5) and no indication for labelling was found. The autoradiogram after SDS–PAGE of the [ 35 S]methionine labelling shows E1A synthesis (4).
Figure Legend Snippet: Fig. 3. Acetylation of c-Jun in vivo . c-Jun was immunoprecipitated with the H79 c-Jun antibody from Ad5HER cells. The cells were mock treated or treated with media supplemented with the acetyl-CoA precursor [ 14 C]pyruvate or [ 35 S]methionine for 1 h. The immunoprecipitations were analysed by a western blot with the Ab-1 anti-c-Jun antibody (1) and labelled immunoprecipitations by autoradiography of [ 35 S]methionine (2) and [ 14 C]pyruvate (3). As a control for the incorporation of 14 C-label in proteins in general, E1A was immunoprecipitated (5) and no indication for labelling was found. The autoradiogram after SDS–PAGE of the [ 35 S]methionine labelling shows E1A synthesis (4).

Techniques Used: In Vivo, Immunoprecipitation, Western Blot, Autoradiography, SDS Page

42) Product Images from "Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1"

Article Title: Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1

Journal: The EMBO Journal

doi: 10.1093/emboj/20.13.3459

Fig. 4. Pin1 binds to c-Jun phosphorylated on Ser 63/73 -Pro. ( A and B ) Modulation of c-Jun phosphorylation by Ras or JNK. HeLa cells were co-transfected with c-Jun or c-Jun S63/73A and Ha-Ras, DN-Ras, activated JNK or control vector. Cells were harvested and cellular proteins were subjected to immunoblotting analysis with antibodies against c-Jun (A) or phosphorylated Ser 63/73 -c-Jun (B). ( C and D ) Interaction between Pin1 and c-Jun phosphorylated on Ser 63/73 -Pro. The same cellular proteins as those described in (A) were incubated with GST–agarose beads that had been pre-incubated with either GST alone or GST–Pin1. Proteins associated with the beads were subjected to immunoblotting analysis with antibodies against c-Jun (C) or phosphorylated Ser 63/73 ). ( E and F ) No interaction between Pin1 and c-Jun S63/73A . The same cellular proteins as those described in the (A) were incubated with GST–agarose beads containing GST or GST–Pin1, and bound proteins were subjected to immunoblotting analysis with antibodies against c-Jun (E) or phosphorylated Ser 63/73 -c-Jun (F). ( G and H ) Co-immunoprecipitation of transfected (G) or endogenous (H) c-Jun with endogenous Pin1. HeLa cells were co-transfected with c-Jun and Ha-Ras or JNK. c-Jun was immunoprecipitated from transfected HeLa cells (G) or non-transfected breast cancer cell lines (H) with polyclonal c-Jun antibodies or non-related antibodies (Control), and then subjected to immunoblotting using monoclonal anti-c-Jun antibodies (upper panel) or anti-Pin1 antibodies (lower panel).
Figure Legend Snippet: Fig. 4. Pin1 binds to c-Jun phosphorylated on Ser 63/73 -Pro. ( A and B ) Modulation of c-Jun phosphorylation by Ras or JNK. HeLa cells were co-transfected with c-Jun or c-Jun S63/73A and Ha-Ras, DN-Ras, activated JNK or control vector. Cells were harvested and cellular proteins were subjected to immunoblotting analysis with antibodies against c-Jun (A) or phosphorylated Ser 63/73 -c-Jun (B). ( C and D ) Interaction between Pin1 and c-Jun phosphorylated on Ser 63/73 -Pro. The same cellular proteins as those described in (A) were incubated with GST–agarose beads that had been pre-incubated with either GST alone or GST–Pin1. Proteins associated with the beads were subjected to immunoblotting analysis with antibodies against c-Jun (C) or phosphorylated Ser 63/73 ). ( E and F ) No interaction between Pin1 and c-Jun S63/73A . The same cellular proteins as those described in the (A) were incubated with GST–agarose beads containing GST or GST–Pin1, and bound proteins were subjected to immunoblotting analysis with antibodies against c-Jun (E) or phosphorylated Ser 63/73 -c-Jun (F). ( G and H ) Co-immunoprecipitation of transfected (G) or endogenous (H) c-Jun with endogenous Pin1. HeLa cells were co-transfected with c-Jun and Ha-Ras or JNK. c-Jun was immunoprecipitated from transfected HeLa cells (G) or non-transfected breast cancer cell lines (H) with polyclonal c-Jun antibodies or non-related antibodies (Control), and then subjected to immunoblotting using monoclonal anti-c-Jun antibodies (upper panel) or anti-Pin1 antibodies (lower panel).

Techniques Used: Transfection, Plasmid Preparation, Incubation, Immunoprecipitation

43) Product Images from "P65 inactivation in adipocytes and macrophages attenuates adipose inflammatory response in lean but not in obese mice"

Article Title: P65 inactivation in adipocytes and macrophages attenuates adipose inflammatory response in lean but not in obese mice

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00532.2014

JNK activity. Phosphorylation of c-Jun was determined in epididymal fat of F-p65-KO mice on HFD (18 wk) in Western blot.
Figure Legend Snippet: JNK activity. Phosphorylation of c-Jun was determined in epididymal fat of F-p65-KO mice on HFD (18 wk) in Western blot.

Techniques Used: Activity Assay, Mouse Assay, Western Blot

44) Product Images from "Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice"

Article Title: Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice

Journal: The Journal of International Medical Research

doi: 10.1177/0300060518761505

Western blotting analysis of the levels of proteins regulating the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways in BGC-823 tumours established in nude mice following approximately 6 weeks of treatment with CP (200 µg/kg body weight) or 0.16% NaHCO3 vehicle control. (a) After 6 weeks of CP and control treatments, levels of the phosphorylated and unphosphorylated forms of AKT, p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected in tumour samples collected from each of the experimental groups by Western blotting analysis (six mice/group). Representative immunoblots are shown. (b) Mean ± SEM relative levels of p-AKT, p-p38, p-JNK, and p-ERK were normalized to those of AKT, p38, JNK, and ERK, respectively. * P
Figure Legend Snippet: Western blotting analysis of the levels of proteins regulating the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways in BGC-823 tumours established in nude mice following approximately 6 weeks of treatment with CP (200 µg/kg body weight) or 0.16% NaHCO3 vehicle control. (a) After 6 weeks of CP and control treatments, levels of the phosphorylated and unphosphorylated forms of AKT, p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected in tumour samples collected from each of the experimental groups by Western blotting analysis (six mice/group). Representative immunoblots are shown. (b) Mean ± SEM relative levels of p-AKT, p-p38, p-JNK, and p-ERK were normalized to those of AKT, p38, JNK, and ERK, respectively. * P

Techniques Used: Western Blot, Mouse Assay

45) Product Images from "AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes *"

Article Title: AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.601310

VopS inhibits activation of NFκB, Erk, and JNK signaling. Phosphorylation and degradation of IκBα, Erk1/2, and JNK phosphorylation is blocked by VopS. HEK293T cells were infected at an m.o.i. 10 for the indicated times, and cells
Figure Legend Snippet: VopS inhibits activation of NFκB, Erk, and JNK signaling. Phosphorylation and degradation of IκBα, Erk1/2, and JNK phosphorylation is blocked by VopS. HEK293T cells were infected at an m.o.i. 10 for the indicated times, and cells

Techniques Used: Activation Assay, Infection

46) Product Images from "FosB Regulates Stretch-Induced Expression of Extracellular Matrix Proteins in Smooth Muscle"

Article Title: FosB Regulates Stretch-Induced Expression of Extracellular Matrix Proteins in Smooth Muscle

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2011.08.034

Stretch stimulation activates select members of the AP-1 family of transcription factors. A: Nuclear extracts (NE) prepared from control or stretch-stimulated pBSMCs were used in an EMSA and assessed for binding to a radiolabeled oligonucleotide carrying a consensus AP-1–binding sequence motif. AP-1–DNA complex formation was detected in both basal and stretch-stimulated pBSMCs (lanes 2 and 9), which was competed out by increasing amounts of unlabeled homologous competitor oligonucleotide [wild type (WT), lanes 3 to 5 and 10 to 12] but not by competitor oligonucleotides carrying a mutated AP-1 site (mutant, lanes 6 to 8 and 13 to 15). Lane 1 is the free radiolabeled probe (no nuclear extract added) FP, free probe. B: The AP-1 complex (lanes 2 and 5) was supershifted (SS) in the presence of antibody to phospho-c-Jun (lanes 4 and 7) but not by an isotype-matched IgG control (lanes 3 and 6). Lane 1, free probe. C: Nuclear extracts from pBSMCs subjected to cyclic stretch for 2 hours or not (control) were assayed using a transcription factor ELISA to determine the DNA-binding activity of AP-1 proteins. The graph shows AP-1 binding expressed as a percentage of control for each of seven AP-1 subunits, and data are representative of at least two independent experiments. Increased binding of c-Jun, Fos, and FosB proteins to a consensus AP-1 oligonucleotide was observed in stretch-treated cells relative to controls. D: Protein lysates from pBSMCs (control or stretched for 24 hours) were immunoblotted with the indicated antibodies. E: Lysates from stretched (24 hours) or control pBSMCs were immunoprecipitated with FosB or isotype-matched IgG control antibodies and immunoblotted for c-Jun. An increase in Jun-FosB interaction is observed on stretch. The decrease in mobility of the c-Jun band is likely because of protein phosphorylation, consistent with D . Nonspecific interaction with the heavy chain of IgG in the sepharose beads used for immunoprecipitation (IP) is observed (IgG H ). All data shown are representative of at least three independent experiments.
Figure Legend Snippet: Stretch stimulation activates select members of the AP-1 family of transcription factors. A: Nuclear extracts (NE) prepared from control or stretch-stimulated pBSMCs were used in an EMSA and assessed for binding to a radiolabeled oligonucleotide carrying a consensus AP-1–binding sequence motif. AP-1–DNA complex formation was detected in both basal and stretch-stimulated pBSMCs (lanes 2 and 9), which was competed out by increasing amounts of unlabeled homologous competitor oligonucleotide [wild type (WT), lanes 3 to 5 and 10 to 12] but not by competitor oligonucleotides carrying a mutated AP-1 site (mutant, lanes 6 to 8 and 13 to 15). Lane 1 is the free radiolabeled probe (no nuclear extract added) FP, free probe. B: The AP-1 complex (lanes 2 and 5) was supershifted (SS) in the presence of antibody to phospho-c-Jun (lanes 4 and 7) but not by an isotype-matched IgG control (lanes 3 and 6). Lane 1, free probe. C: Nuclear extracts from pBSMCs subjected to cyclic stretch for 2 hours or not (control) were assayed using a transcription factor ELISA to determine the DNA-binding activity of AP-1 proteins. The graph shows AP-1 binding expressed as a percentage of control for each of seven AP-1 subunits, and data are representative of at least two independent experiments. Increased binding of c-Jun, Fos, and FosB proteins to a consensus AP-1 oligonucleotide was observed in stretch-treated cells relative to controls. D: Protein lysates from pBSMCs (control or stretched for 24 hours) were immunoblotted with the indicated antibodies. E: Lysates from stretched (24 hours) or control pBSMCs were immunoprecipitated with FosB or isotype-matched IgG control antibodies and immunoblotted for c-Jun. An increase in Jun-FosB interaction is observed on stretch. The decrease in mobility of the c-Jun band is likely because of protein phosphorylation, consistent with D . Nonspecific interaction with the heavy chain of IgG in the sepharose beads used for immunoprecipitation (IP) is observed (IgG H ). All data shown are representative of at least three independent experiments.

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunoprecipitation

47) Product Images from "Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis"

Article Title: Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis

Journal: Oncotarget

doi:

CEP-1347 treatment decreases JNK activity and c-Jun phosphorylation in MCF-7 and LCC9 cells Cells were treated with vehicle or 100 nM CEP-1347 for 48 h. Cellular lysates were prepared and analyzed by western blotting using the indicated antibodies. Actin was used as loading control. (A) Levels of phospho-c-Jun in vehicle and CEP-1347 treated cells. Upper : representative blots. Lower : quantitative analysis of p-c-Jun levels, normalized to β-actin levels. Results represent the mean +/− SD of three independent experiments. **p
Figure Legend Snippet: CEP-1347 treatment decreases JNK activity and c-Jun phosphorylation in MCF-7 and LCC9 cells Cells were treated with vehicle or 100 nM CEP-1347 for 48 h. Cellular lysates were prepared and analyzed by western blotting using the indicated antibodies. Actin was used as loading control. (A) Levels of phospho-c-Jun in vehicle and CEP-1347 treated cells. Upper : representative blots. Lower : quantitative analysis of p-c-Jun levels, normalized to β-actin levels. Results represent the mean +/− SD of three independent experiments. **p

Techniques Used: Activity Assay, Western Blot

48) Product Images from "Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1"

Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1200573

ESAT-6 differentially regulates expression and DNA-binding activities of IRF-1 and AP-1 transcription factors A. iDCs from four donors were stimulated with LPS/CD40LT, with or without ESAT-6. Four h later, total cell protein extracts were prepared, and Western blotting was performed to determine expression of IRF-1 and phosphorylation of the AP-1 transcription factors, ATF-2 and c-Jun. The blots were stripped and reblotted for expression of GAPDH as a protein loading control. A representative result is shown. B. iDCs from four donors were matured as in A, and DNA binding activity of the nuclear protein extracts was evaluated by electrophoretic mobility shift assays, using the radiolabeled AP-1 binding site of the p19 promoter as a probe. The DNA:protein complexes were resolved by 5% non-denaturing polyacrilamide gel electrophoresis and visualized by autoradiography. A representative result is shown. C. Nuclear extracts of DCs from four donors, matured with LPS/CD40LT and ESAT-6 were incubated with unlabeled double-stranded DNA or different IgGs, as indicated, for 30 min, followed by incubation with the radiolabeled AP-1 site of p19, and visualized as in B. A representative result is shown. (ATF-2 1 , pAb and ATF-2 2 , mAb) D. Chromatin immunoprecipitation. iDCs from four donors were treated with LPS/CD40LT, with or without ESAT-6, as described in A. Six hrs later, the cells were incubated with 1% formaldehyde, followed by preparation of nuclear chromatin supernatants and immunoprecipitation with anti-IRF-1 or isotype-matched control IgG (c.Ig). The precipitated DNA samples were amplified with primers for a 187 bp DNA fragment that contains the IRF-1 binding site of the p35 promoter. The PCR product was visualized by 1% agarose gel analysis. A representative result is shown.
Figure Legend Snippet: ESAT-6 differentially regulates expression and DNA-binding activities of IRF-1 and AP-1 transcription factors A. iDCs from four donors were stimulated with LPS/CD40LT, with or without ESAT-6. Four h later, total cell protein extracts were prepared, and Western blotting was performed to determine expression of IRF-1 and phosphorylation of the AP-1 transcription factors, ATF-2 and c-Jun. The blots were stripped and reblotted for expression of GAPDH as a protein loading control. A representative result is shown. B. iDCs from four donors were matured as in A, and DNA binding activity of the nuclear protein extracts was evaluated by electrophoretic mobility shift assays, using the radiolabeled AP-1 binding site of the p19 promoter as a probe. The DNA:protein complexes were resolved by 5% non-denaturing polyacrilamide gel electrophoresis and visualized by autoradiography. A representative result is shown. C. Nuclear extracts of DCs from four donors, matured with LPS/CD40LT and ESAT-6 were incubated with unlabeled double-stranded DNA or different IgGs, as indicated, for 30 min, followed by incubation with the radiolabeled AP-1 site of p19, and visualized as in B. A representative result is shown. (ATF-2 1 , pAb and ATF-2 2 , mAb) D. Chromatin immunoprecipitation. iDCs from four donors were treated with LPS/CD40LT, with or without ESAT-6, as described in A. Six hrs later, the cells were incubated with 1% formaldehyde, followed by preparation of nuclear chromatin supernatants and immunoprecipitation with anti-IRF-1 or isotype-matched control IgG (c.Ig). The precipitated DNA samples were amplified with primers for a 187 bp DNA fragment that contains the IRF-1 binding site of the p35 promoter. The PCR product was visualized by 1% agarose gel analysis. A representative result is shown.

Techniques Used: Expressing, Binding Assay, Western Blot, Activity Assay, Electrophoretic Mobility Shift Assay, Nucleic Acid Electrophoresis, Autoradiography, Incubation, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

49) Product Images from "Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase C?-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1"

Article Title: Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase C?-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2010.00762.x

Effect of metformin on intracellular Ca 2+ concentration in HT-1080 cells. (A) Cells (2 × 10 5 cells·mL −1 n a 24-well microtitre plate) were treated with PMA (30 nM) for 30 min in the presence or absence of metformin, BAPTA-AM or EGTA, or 30 min at 37°C in a humidified incubator with a 5% CO 2 atmosphere. Cells were observed under a fluorescence microscope (excitation at 385 nm; emission at 512 nm). Each figure is representative of at least three others. (B) Effect of the calcium chelator BAPTA-AM or Ca 2+ /CaM antagonist W7 on PMA-induced phosphorylation of CAMKI, PKCα, ERK and JNK in HT-1080 cells. Cells were treated with PMA (30 nM) for 30 min in the presence or absence of BAPTA-AM or W7, and the phosphorylation levels of CAMKI, PKCα, ERK and JNK were measured by Western blotting. Each blot is representative of at least three others. (C) Effect of the calcium chelator BAPTA-AM or Ca 2+ /CaM antagonist W7 on PMA-induced c-Jun and c-Fos translocation in HT-1080 cells. Cells were pretreated with BAPTA-AM (10 µM) or W7 (40 µM) for 1 h, and treated with 30 nM PMA for 3 h. Nuclear extracts were subjected to SDS–PAGE followed by Western blotting with anti-c-Jun, anti-c-Fos and anti-lamin B antibodies. Each blot is representative of at least three others. (D and E) Effect of BAPTA-AM or W7 on PMA-induced MMP-9 activity in HT-1080 cells. Cells were pretreated with BAPTA-AM (10 µM) or W7 (40 µM) for 1 h, and treated with 30 nM PMA for 24 h. MMP-9 activity in the medium was analysed by gelatin zymography and Western blotting. Each blot is representative of at least three others. Cells were transfected with pGL-MMP-9WT reporter plasmids. The luciferase activity in the cell extract was determined. Data are expressed as the means ± SD of triplicate experiments. *Significantly different from PMA treatment only ( P
Figure Legend Snippet: Effect of metformin on intracellular Ca 2+ concentration in HT-1080 cells. (A) Cells (2 × 10 5 cells·mL −1 n a 24-well microtitre plate) were treated with PMA (30 nM) for 30 min in the presence or absence of metformin, BAPTA-AM or EGTA, or 30 min at 37°C in a humidified incubator with a 5% CO 2 atmosphere. Cells were observed under a fluorescence microscope (excitation at 385 nm; emission at 512 nm). Each figure is representative of at least three others. (B) Effect of the calcium chelator BAPTA-AM or Ca 2+ /CaM antagonist W7 on PMA-induced phosphorylation of CAMKI, PKCα, ERK and JNK in HT-1080 cells. Cells were treated with PMA (30 nM) for 30 min in the presence or absence of BAPTA-AM or W7, and the phosphorylation levels of CAMKI, PKCα, ERK and JNK were measured by Western blotting. Each blot is representative of at least three others. (C) Effect of the calcium chelator BAPTA-AM or Ca 2+ /CaM antagonist W7 on PMA-induced c-Jun and c-Fos translocation in HT-1080 cells. Cells were pretreated with BAPTA-AM (10 µM) or W7 (40 µM) for 1 h, and treated with 30 nM PMA for 3 h. Nuclear extracts were subjected to SDS–PAGE followed by Western blotting with anti-c-Jun, anti-c-Fos and anti-lamin B antibodies. Each blot is representative of at least three others. (D and E) Effect of BAPTA-AM or W7 on PMA-induced MMP-9 activity in HT-1080 cells. Cells were pretreated with BAPTA-AM (10 µM) or W7 (40 µM) for 1 h, and treated with 30 nM PMA for 24 h. MMP-9 activity in the medium was analysed by gelatin zymography and Western blotting. Each blot is representative of at least three others. Cells were transfected with pGL-MMP-9WT reporter plasmids. The luciferase activity in the cell extract was determined. Data are expressed as the means ± SD of triplicate experiments. *Significantly different from PMA treatment only ( P

Techniques Used: Concentration Assay, Fluorescence, Microscopy, Chick Chorioallantoic Membrane Assay, Western Blot, Translocation Assay, SDS Page, Activity Assay, Zymography, Transfection, Luciferase

50) Product Images from "Enhancer RNA and NFκB-dependent P300 regulation of ADAMDEC1"

Article Title: Enhancer RNA and NFκB-dependent P300 regulation of ADAMDEC1

Journal: Molecular immunology

doi: 10.1016/j.molimm.2018.09.019

ChIP-seq of the enhancer regions MonoMac6 cells were treated with 1μg/ml LPS for 90 minutes. ChIP assays were performed with antibodies H3K4me3, H3K27ac, c-JUN and P65. In resting cells, H3K4me3 was highest at Enhancer 1. LPS stimulation let to increased c-JUN at Enhancer 1, ADAM28 promoter, H3K27ac at the A DAMDEC1 promoter and Enhancer 2 as well as binding of p65 to the ADAM28 promoter and Enhancer 2. (n=4, error bars represent SD, * indicates p
Figure Legend Snippet: ChIP-seq of the enhancer regions MonoMac6 cells were treated with 1μg/ml LPS for 90 minutes. ChIP assays were performed with antibodies H3K4me3, H3K27ac, c-JUN and P65. In resting cells, H3K4me3 was highest at Enhancer 1. LPS stimulation let to increased c-JUN at Enhancer 1, ADAM28 promoter, H3K27ac at the A DAMDEC1 promoter and Enhancer 2 as well as binding of p65 to the ADAM28 promoter and Enhancer 2. (n=4, error bars represent SD, * indicates p

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

51) Product Images from "The transcription factor c-Fos coordinates with histone lysine-specific demethylase 2A to activate the expression of cyclooxygenase-2"

Article Title: The transcription factor c-Fos coordinates with histone lysine-specific demethylase 2A to activate the expression of cyclooxygenase-2

Journal: Oncotarget

doi:

c-Fos interacts with KDM2A in vitro and in vivo A. A Myc-tagged c-Fos and a Flag-tagged histone modification enzyme (as indicated in the figure) were co-transfected into HEK293T cells and the proteins were then extracted for Co-IP with anti-Myc and anti-Flag antibodies, followed by immunoblotting with anti-Myc. B. GST-KDM2A was incubated with His-c-Fos, and Western blotting or Coomassie staining was performed to detect the direct binding of c-Fos and KDM2A in vitro . * indicates the specific bands. C. Schematic of plasmids encoding full-length c-Fos, an N-terminal fragment (aa 1–136), a middle fragment (aa 137–200) and a C-terminal fragment (aa 201–381). D. His-c-Fos FL or fragments were incubated with GST-KDM2A, and Western blotting with an anti-GST antibody or Coomassie staining was performed to detect the interaction. # indicates the specific bands. E. H719 cells were treated without or with TPA for 2 hrs at 100 ng/mL, then the cell were extracted for Co-IP using an anti-c-Fos antibody, followed by Western blotting using anti-KDM2A or anti-c-Fos antibodies to detect the interaction between c-Fos and KDM2A. F. H719 cells were treated without or with TPA for 2 hrs at 100 ng/mL, then the cells were extracted for Co-IP using anti-KDM2A antibody, followed by Western blotting using an anti-KDM2A or anti-c-Fos antibodies to detect the interaction between c-Fos and KDM2A. G. HEK293T cells were transfected with pECFP-c-Fos and/or pEYFP-KDM2A plasmids, followed by TPA treatment for 2 hrs. FLIM assays were performed, and the images of representative cells are shown for each group. H. FRET efficiency is shown in the column diagram, and the representative value is an average FRET efficiency of 23 cells with standard error. **, p
Figure Legend Snippet: c-Fos interacts with KDM2A in vitro and in vivo A. A Myc-tagged c-Fos and a Flag-tagged histone modification enzyme (as indicated in the figure) were co-transfected into HEK293T cells and the proteins were then extracted for Co-IP with anti-Myc and anti-Flag antibodies, followed by immunoblotting with anti-Myc. B. GST-KDM2A was incubated with His-c-Fos, and Western blotting or Coomassie staining was performed to detect the direct binding of c-Fos and KDM2A in vitro . * indicates the specific bands. C. Schematic of plasmids encoding full-length c-Fos, an N-terminal fragment (aa 1–136), a middle fragment (aa 137–200) and a C-terminal fragment (aa 201–381). D. His-c-Fos FL or fragments were incubated with GST-KDM2A, and Western blotting with an anti-GST antibody or Coomassie staining was performed to detect the interaction. # indicates the specific bands. E. H719 cells were treated without or with TPA for 2 hrs at 100 ng/mL, then the cell were extracted for Co-IP using an anti-c-Fos antibody, followed by Western blotting using anti-KDM2A or anti-c-Fos antibodies to detect the interaction between c-Fos and KDM2A. F. H719 cells were treated without or with TPA for 2 hrs at 100 ng/mL, then the cells were extracted for Co-IP using anti-KDM2A antibody, followed by Western blotting using an anti-KDM2A or anti-c-Fos antibodies to detect the interaction between c-Fos and KDM2A. G. HEK293T cells were transfected with pECFP-c-Fos and/or pEYFP-KDM2A plasmids, followed by TPA treatment for 2 hrs. FLIM assays were performed, and the images of representative cells are shown for each group. H. FRET efficiency is shown in the column diagram, and the representative value is an average FRET efficiency of 23 cells with standard error. **, p

Techniques Used: In Vitro, In Vivo, Modification, Transfection, Co-Immunoprecipitation Assay, Incubation, Western Blot, Staining, Binding Assay

52) Product Images from "Epstein-Barr Virus Latent Membrane Protein 2A Regulates c-Jun Protein through Extracellular Signal-Regulated Kinase"

Article Title: Epstein-Barr Virus Latent Membrane Protein 2A Regulates c-Jun Protein through Extracellular Signal-Regulated Kinase

Journal: Journal of Virology

doi: 10.1128/JVI.76.18.9556-9561.2002

Investigation of the phosphorylation status of MAPK downstream effectors in LMP2A-expressing clones. (A) Antibodies that specifically react with phospho-c-Jun were used to investigate the phosphorylation of c-Jun in stably expressing clones (stable) and transient transfectants (trans) in LMP2A-expressing (2A) and vector control (V) cells. UV-treated (200 J/m 2 ) 293 cells (UV) served as the positive controls for phospho-c-Jun induction. (B) Antibodies that specifically react with phospho-ATF2 were used to investigate the phosphorylation of ATF2 in LMP2A-expressing (2A) and vector control (V) cells. The detection of tubulin served as an internal control of protein amounts. The numbers to the left of each panel represent molecular mass in kilodaltons.
Figure Legend Snippet: Investigation of the phosphorylation status of MAPK downstream effectors in LMP2A-expressing clones. (A) Antibodies that specifically react with phospho-c-Jun were used to investigate the phosphorylation of c-Jun in stably expressing clones (stable) and transient transfectants (trans) in LMP2A-expressing (2A) and vector control (V) cells. UV-treated (200 J/m 2 ) 293 cells (UV) served as the positive controls for phospho-c-Jun induction. (B) Antibodies that specifically react with phospho-ATF2 were used to investigate the phosphorylation of ATF2 in LMP2A-expressing (2A) and vector control (V) cells. The detection of tubulin served as an internal control of protein amounts. The numbers to the left of each panel represent molecular mass in kilodaltons.

Techniques Used: Expressing, Stable Transfection, Plasmid Preparation

53) Product Images from "Members of the AP-1 Family, c-Jun and c-Fos, Functionally Interact with JC Virus Early Regulatory Protein Large T Antigen"

Article Title: Members of the AP-1 Family, c-Jun and c-Fos, Functionally Interact with JC Virus Early Regulatory Protein Large T Antigen

Journal: Journal of Virology

doi: 10.1128/JVI.77.9.5241-5252.2003

T-Ag inhibits c-Jun binding to its target sequences in an electrophoretic mobility shift assay. (A) Band shift assay. A double-stranded synthetic oligonucleotide containing JCV AP-1 binding site (5′-CAAGCATGAGCTCATACCTA-3′) spanning nucleotides 155 to 162 of JCV Mad-1 regulatory region was end labeled with [γ- 32 P]ATP with T4 polynucleotide kinase and gel purified. Nuclear extracts (10 μg/lane) prepared from U-87MG cells and untransfected (lane 2) or transfected with a T-Ag expression plasmid (lane 3) were incubated with labeled probe (40,000 cpm/lane) in a binding buffer, as described in Materials and Methods. In addition, probe plus nuclear extract from T-Ag-transfected cells was also incubated with either unlabeled wild-type oligonucleotide (WT) (lanes 4 and 5) or 25- or 150-fold molar excesses of its mutant variant competitor oligonucleotides (Mut) (5′-CAAGCAT T AGCT TG TACCTA-3′; bold indicates base substitutions relative to the wild type) (lanes 6 and 7, respectively). Probe plus nuclear extract mixture was also incubated either with a preimmune (α-pre; 2 μg) (lane 8) or an anti-c-Jun (α-c-Jun, KM-1; 2 μg) (lane 9) antibody. Formed DNA-protein complexes were then resolved on a 6% polyacrylamide gel under native conditions and visualized by autoradiography. The specific DNA-protein complexes are indicated by an arrow, and nonspecific complexes are indicated by a solid arrowhead. A bracket indicates antibody supershifted complexes. (B) Western blot analysis. Nuclear extracts prepared from either untransfected U-87MG cells (lane 1) or U-87MG cells transfected with CMV-T-Ag expression plasmid (lane 2) were analyzed by Western blotting using an anti-SV40 T-Ag antibody (Ab-2 416) which is cross-reactive with JCV T-Ag. Comp, competitor; Ab, antibody; Tfxn, transfection.
Figure Legend Snippet: T-Ag inhibits c-Jun binding to its target sequences in an electrophoretic mobility shift assay. (A) Band shift assay. A double-stranded synthetic oligonucleotide containing JCV AP-1 binding site (5′-CAAGCATGAGCTCATACCTA-3′) spanning nucleotides 155 to 162 of JCV Mad-1 regulatory region was end labeled with [γ- 32 P]ATP with T4 polynucleotide kinase and gel purified. Nuclear extracts (10 μg/lane) prepared from U-87MG cells and untransfected (lane 2) or transfected with a T-Ag expression plasmid (lane 3) were incubated with labeled probe (40,000 cpm/lane) in a binding buffer, as described in Materials and Methods. In addition, probe plus nuclear extract from T-Ag-transfected cells was also incubated with either unlabeled wild-type oligonucleotide (WT) (lanes 4 and 5) or 25- or 150-fold molar excesses of its mutant variant competitor oligonucleotides (Mut) (5′-CAAGCAT T AGCT TG TACCTA-3′; bold indicates base substitutions relative to the wild type) (lanes 6 and 7, respectively). Probe plus nuclear extract mixture was also incubated either with a preimmune (α-pre; 2 μg) (lane 8) or an anti-c-Jun (α-c-Jun, KM-1; 2 μg) (lane 9) antibody. Formed DNA-protein complexes were then resolved on a 6% polyacrylamide gel under native conditions and visualized by autoradiography. The specific DNA-protein complexes are indicated by an arrow, and nonspecific complexes are indicated by a solid arrowhead. A bracket indicates antibody supershifted complexes. (B) Western blot analysis. Nuclear extracts prepared from either untransfected U-87MG cells (lane 1) or U-87MG cells transfected with CMV-T-Ag expression plasmid (lane 2) were analyzed by Western blotting using an anti-SV40 T-Ag antibody (Ab-2 416) which is cross-reactive with JCV T-Ag. Comp, competitor; Ab, antibody; Tfxn, transfection.

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Purification, Transfection, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Variant Assay, Autoradiography, Western Blot

54) Product Images from "Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression"

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600437

Differential regulation of PPARβ expression of in vitro model mimicking different stages of wound repair. ( A, B ) RPA analyses of PPARβ, keratin 5 (K5) and 6 (K6) after exposure of the keratinocytes to various treatments, as indicated. NM: necrosis-derived conditioned medium mimicking early inflammation, initiated by using either minced skin (MS) or freezed/thawed fibroblasts (FT); AM: apoptotic-derived conditioned medium mimicking the context of late remodeling stages of wound healing, initiated by using either UV (UV)- or dexamethasone (Dex)-treated fibroblasts. The importance of TGF-β1 in AM was assessed by preincubation of AM with anti-TGF-β1 antibody (50 μg/ml). TGF-β1 (5 ng/ml), TNF-α (5 ng/ml) and LPS (1 ng/ml) were used. In all RPA analyses, data were normalized using the ribosomal protein L27 mRNA levels. Values represent the mean of at least three independent experiments. ( C ) ChIP performed on primary keratinocytes treated as in (B) with AM or NM in the presence of TNF-α or TGF-β1, as indicated. The results show a PCR amplification of the AP-1(−414) site (right panel) or control sequence (left panel) after chIP with anti-c-JUN antibody. Preimmune serum serves as a control for chIP. ( D ) JunB is not binding to the AP-1 site of the PPARβ promoter. ChIP assays were performed with the anti-JunB antibody. Keratinocytes exposed to NM, in the absence or presence of TGF-β1, were used for chIP. The AP-1(−414) site of the PPARβ promoter could not be amplified, indicating the lack of JunB binding to this site (top panel). As a positive control, the AP-1 site at position −783 of the mouse p16 INK4a gene was amplified using senescent (passages 6 and 12) primary fibroblasts (bottom panel). For (C) and (D), the figure shows a representative result out of four independent experiments.
Figure Legend Snippet: Differential regulation of PPARβ expression of in vitro model mimicking different stages of wound repair. ( A, B ) RPA analyses of PPARβ, keratin 5 (K5) and 6 (K6) after exposure of the keratinocytes to various treatments, as indicated. NM: necrosis-derived conditioned medium mimicking early inflammation, initiated by using either minced skin (MS) or freezed/thawed fibroblasts (FT); AM: apoptotic-derived conditioned medium mimicking the context of late remodeling stages of wound healing, initiated by using either UV (UV)- or dexamethasone (Dex)-treated fibroblasts. The importance of TGF-β1 in AM was assessed by preincubation of AM with anti-TGF-β1 antibody (50 μg/ml). TGF-β1 (5 ng/ml), TNF-α (5 ng/ml) and LPS (1 ng/ml) were used. In all RPA analyses, data were normalized using the ribosomal protein L27 mRNA levels. Values represent the mean of at least three independent experiments. ( C ) ChIP performed on primary keratinocytes treated as in (B) with AM or NM in the presence of TNF-α or TGF-β1, as indicated. The results show a PCR amplification of the AP-1(−414) site (right panel) or control sequence (left panel) after chIP with anti-c-JUN antibody. Preimmune serum serves as a control for chIP. ( D ) JunB is not binding to the AP-1 site of the PPARβ promoter. ChIP assays were performed with the anti-JunB antibody. Keratinocytes exposed to NM, in the absence or presence of TGF-β1, were used for chIP. The AP-1(−414) site of the PPARβ promoter could not be amplified, indicating the lack of JunB binding to this site (top panel). As a positive control, the AP-1 site at position −783 of the mouse p16 INK4a gene was amplified using senescent (passages 6 and 12) primary fibroblasts (bottom panel). For (C) and (D), the figure shows a representative result out of four independent experiments.

Techniques Used: Expressing, In Vitro, Recombinase Polymerase Amplification, Derivative Assay, Mass Spectrometry, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Binding Assay, Positive Control

Effects of Smad3–c-JUN interaction are promoter context dependent. ( A ) Cotransfection of the PPARβ(−445) promoter construct with increasing amounts of Smad3, Smad3(4A) (left panel), SmadΔ3-NLS (middle panel) and Smad3ΔC (right panel). Similar expression levels of Smad3 and Smad3(4A) were verified by immunoblotting with anti-FLAG antibody, whereas SmadΔ3-NLS and Smad3ΔC were verified by anti-myc antibody (see top of each panel); V: empty expression vector. The means of at least six independent experiments are shown. ( B ) Smad3–c-JUN interaction inhibits PPARβ expression. Protein–protein crosslink and chIP with anti-c-JUN (top panel) and anti-p300 (middle panel) antibodies, followed by Western blot (WB) with anti-Smad3, anti-Smad4 antibody or PCR amplification of the AP-1(−414) site on the PPARβ promoter are shown. In re-chIP experiment (bottom panel), first chIP was performed with anti-c-JUN antibody. After dissociation from the anti-c-JUN antibody, a second chIP (or re-chIP) was performed with anti-p300 antibody. Prior chIP, equal input was verified by immunoblotting with anti-tubulin antibody. A representative result out of three independent experiments is shown. ( C ) p300–c-Jun and p300–Smad3 complexes stimulate human PAI expression. ChIPs were performed on keratinocytes transfected with a 800 bp hPAI luciferase promoter construct and coincubated with TNF-α, AM, NM or TGF-β1 as indicated. ChIP assays with anti-c-JUN (top panel), chIP/re-chIP with anti-c-JUN/anti-p300 (middle panel) and anti-FLAG/anti-p300 (bottom panel) antibodies are shown. Our results also suggest that both transcription factors can recruit p300 coactivator to the hPAI promoter in response to either TNF-α/NM or TGF-β1/AM. One representative result out of three independent experiments is shown.
Figure Legend Snippet: Effects of Smad3–c-JUN interaction are promoter context dependent. ( A ) Cotransfection of the PPARβ(−445) promoter construct with increasing amounts of Smad3, Smad3(4A) (left panel), SmadΔ3-NLS (middle panel) and Smad3ΔC (right panel). Similar expression levels of Smad3 and Smad3(4A) were verified by immunoblotting with anti-FLAG antibody, whereas SmadΔ3-NLS and Smad3ΔC were verified by anti-myc antibody (see top of each panel); V: empty expression vector. The means of at least six independent experiments are shown. ( B ) Smad3–c-JUN interaction inhibits PPARβ expression. Protein–protein crosslink and chIP with anti-c-JUN (top panel) and anti-p300 (middle panel) antibodies, followed by Western blot (WB) with anti-Smad3, anti-Smad4 antibody or PCR amplification of the AP-1(−414) site on the PPARβ promoter are shown. In re-chIP experiment (bottom panel), first chIP was performed with anti-c-JUN antibody. After dissociation from the anti-c-JUN antibody, a second chIP (or re-chIP) was performed with anti-p300 antibody. Prior chIP, equal input was verified by immunoblotting with anti-tubulin antibody. A representative result out of three independent experiments is shown. ( C ) p300–c-Jun and p300–Smad3 complexes stimulate human PAI expression. ChIPs were performed on keratinocytes transfected with a 800 bp hPAI luciferase promoter construct and coincubated with TNF-α, AM, NM or TGF-β1 as indicated. ChIP assays with anti-c-JUN (top panel), chIP/re-chIP with anti-c-JUN/anti-p300 (middle panel) and anti-FLAG/anti-p300 (bottom panel) antibodies are shown. Our results also suggest that both transcription factors can recruit p300 coactivator to the hPAI promoter in response to either TNF-α/NM or TGF-β1/AM. One representative result out of three independent experiments is shown.

Techniques Used: Cotransfection, Construct, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Western Blot, Polymerase Chain Reaction, Amplification, Transfection, Luciferase

Smad3 is essential for inhibition of inflammation-induced PPARβ expression and downregulation of its target genes. ( A ) RPA analysis of PPARβ mRNA expression after treatment of Smad3 +/+ and Smad3 −/− primary keratinocytes with TNF-α (5 ng/ml) in the presence or absence of TGF-β1 (5 ng/ml). Ribosomal protein (L27) mRNA was used as internal control. Vehicle (PBS)-treated keratinocytes served as control. ( B ) RPA analyses of PPARβ, ILK and PDK1 mRNA expression in keratinocytes exposed to day 1 wound fluid (WFD1; 0.1 and 0.5% v/v) in the presence or absence of day 7 wound fluid (WFD7; 0.1 and 0.5% v/v). The importance of TGF-β1 in WFD7 was assessed by preincubation of WFD7 with anti-TGF-β1 antibody (10 and 50 μg/ml). ChIP analysis with anti-c-JUN antibody confirmed reduced c-JUN binding to AP-1(−414) of the PPARβ promoter in the presence of untreated or control Ig-treated (10 and 50 μg/ml) WFD7. The means of at least four independent experiments are shown. ( C ) RPA analyses of expression levels of PPARβ, ILK and PDK1 in day 1 (inflammation phase) and 7 (re-epithelialization phase) wound biopsies from Smad3 +/+ and Smad3 −/− mice, normalized against ribosomal protein (L27) mRNA levels. The experimental value, from unwounded wild-type skin, used as normalization unit was arbitrarily assigned a value of 1. The other values indicate relative fold increase (decrease) as compared to unwounded wild-type skin. Values represent the mean of three independent experiments. ( D ). Hence, during early phases of wound repair, the growth–survival Akt1 pathway is dominant. As wound repair proceeds into the re-epithelialization/remodeling phase, TGF-β1 produced by both the infiltrating immune cells and the wound fibroblasts leads to strong activation of the TGF-β1/Smad3 pathway. Phosphorylation of Smad3 prevents the formation of Akt1–Smad3 complex, forms a complex with Smad4, which translocates to the nucleus and regulates gene expression. Smad3 either interacts with c-JUN or sequesters the p300 coactivator, which inhibits inflammation-induced PPARβ expression. Reduced Akt1 phosphorylation amplifies the effect of Smad3. Importantly, this mechanism allows for a shift in the control of PPARβ and downstream effects, from the prevalence of growth–survival pathway to the prevalence of TGF-β1-mediated growth arrest.
Figure Legend Snippet: Smad3 is essential for inhibition of inflammation-induced PPARβ expression and downregulation of its target genes. ( A ) RPA analysis of PPARβ mRNA expression after treatment of Smad3 +/+ and Smad3 −/− primary keratinocytes with TNF-α (5 ng/ml) in the presence or absence of TGF-β1 (5 ng/ml). Ribosomal protein (L27) mRNA was used as internal control. Vehicle (PBS)-treated keratinocytes served as control. ( B ) RPA analyses of PPARβ, ILK and PDK1 mRNA expression in keratinocytes exposed to day 1 wound fluid (WFD1; 0.1 and 0.5% v/v) in the presence or absence of day 7 wound fluid (WFD7; 0.1 and 0.5% v/v). The importance of TGF-β1 in WFD7 was assessed by preincubation of WFD7 with anti-TGF-β1 antibody (10 and 50 μg/ml). ChIP analysis with anti-c-JUN antibody confirmed reduced c-JUN binding to AP-1(−414) of the PPARβ promoter in the presence of untreated or control Ig-treated (10 and 50 μg/ml) WFD7. The means of at least four independent experiments are shown. ( C ) RPA analyses of expression levels of PPARβ, ILK and PDK1 in day 1 (inflammation phase) and 7 (re-epithelialization phase) wound biopsies from Smad3 +/+ and Smad3 −/− mice, normalized against ribosomal protein (L27) mRNA levels. The experimental value, from unwounded wild-type skin, used as normalization unit was arbitrarily assigned a value of 1. The other values indicate relative fold increase (decrease) as compared to unwounded wild-type skin. Values represent the mean of three independent experiments. ( D ). Hence, during early phases of wound repair, the growth–survival Akt1 pathway is dominant. As wound repair proceeds into the re-epithelialization/remodeling phase, TGF-β1 produced by both the infiltrating immune cells and the wound fibroblasts leads to strong activation of the TGF-β1/Smad3 pathway. Phosphorylation of Smad3 prevents the formation of Akt1–Smad3 complex, forms a complex with Smad4, which translocates to the nucleus and regulates gene expression. Smad3 either interacts with c-JUN or sequesters the p300 coactivator, which inhibits inflammation-induced PPARβ expression. Reduced Akt1 phosphorylation amplifies the effect of Smad3. Importantly, this mechanism allows for a shift in the control of PPARβ and downstream effects, from the prevalence of growth–survival pathway to the prevalence of TGF-β1-mediated growth arrest.

Techniques Used: Inhibition, Expressing, Recombinase Polymerase Amplification, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay, Produced, Activation Assay

55) Product Images from "Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †"

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01282-09

Mechanisms of inhibition of IL-2 expression by VIP. (A) Human T cells were cultured with medium (unstim) or CD3/CD28 activated without or with VIP (10 −7 M). After 4 h, nuclear proteins were isolated and assayed for DNA binding to NF-κB, AP-1, or NFAT consensus sites by EMSA and expressed as densitometric units relative to the binding of the constitutive nuclear factor NF-Y. The error bars indicate SD. (B) After 3 h, whole-cell extracts were analyzed by Western blotting for c-Jun and phospho-c-Jun expression. NFATp levels were determined in both nuclear and cytoplasmic extracts and expressed as densitometric units relative to laminin B or β-tubulin. (C) After 1 h of culture, cell lysates were prepared, and activation of ERK1/2 and MEK1 was determined by Western blotting with phosphospecific Abs and expressed as densitometric units normalized for total ERK1/2 or MEK1. The kinase activity of Raf1 was determined by assaying the phosphotransferase activity toward GST-MEK1. (D) Activation of Rap1 and Ras was determined as described in Materials and Methods. The inactive forms of Raf1 were measured in cell lysates by Western blotting for phospho-Ser 43 -Raf1 and phospho-Ser 259 -Raf1. n = 3 or 4. *, P
Figure Legend Snippet: Mechanisms of inhibition of IL-2 expression by VIP. (A) Human T cells were cultured with medium (unstim) or CD3/CD28 activated without or with VIP (10 −7 M). After 4 h, nuclear proteins were isolated and assayed for DNA binding to NF-κB, AP-1, or NFAT consensus sites by EMSA and expressed as densitometric units relative to the binding of the constitutive nuclear factor NF-Y. The error bars indicate SD. (B) After 3 h, whole-cell extracts were analyzed by Western blotting for c-Jun and phospho-c-Jun expression. NFATp levels were determined in both nuclear and cytoplasmic extracts and expressed as densitometric units relative to laminin B or β-tubulin. (C) After 1 h of culture, cell lysates were prepared, and activation of ERK1/2 and MEK1 was determined by Western blotting with phosphospecific Abs and expressed as densitometric units normalized for total ERK1/2 or MEK1. The kinase activity of Raf1 was determined by assaying the phosphotransferase activity toward GST-MEK1. (D) Activation of Rap1 and Ras was determined as described in Materials and Methods. The inactive forms of Raf1 were measured in cell lysates by Western blotting for phospho-Ser 43 -Raf1 and phospho-Ser 259 -Raf1. n = 3 or 4. *, P

Techniques Used: Inhibition, Expressing, Cell Culture, Isolation, Binding Assay, Western Blot, Activation Assay, Activity Assay

56) Product Images from "The AP-1 transcription factor JunB is required for Th17 cell differentiation"

Article Title: The AP-1 transcription factor JunB is required for Th17 cell differentiation

Journal: Scientific Reports

doi: 10.1038/s41598-017-17597-3

JunB but not c-Jun is abundantly expressed in Th17 cells. ( A ) Immunoblot analysis for evaluation of relative expression levels of endogenous Jun family proteins in Th17 cells. The same amounts of FLAG–JunB, FLAG–c-Jun or FLAG–JunD, which were expressed in HEK293T cells, were estimated by immunoblot with an anti-FLAG antibody (M2) (left panel). Serially diluted proteins and the Th17 cell lysate were subjected to immunoblot analysis with anti-JunB, anti-c-Jun, or anti-JunD antibodies (middle panel), followed by quantification with Odyssey Infrared Imaging System. FLAG-tagged and endogenous proteins were denoted by white and black arrowheads, respectively. Relative protein levels of endogenous JunB, c-Jun, and JunD were shown in the right panel in (A) . ( B ) Real-time PCR analysis for relative mRNA copy numbers of Junb and Jun in naive CD4 + T cells and Th17 cells. mRNA copy numbers were estimated from standard curves that were generated using known numbers of a plasmid encoding Junb or Jun . Data are presented as mean ± SD.
Figure Legend Snippet: JunB but not c-Jun is abundantly expressed in Th17 cells. ( A ) Immunoblot analysis for evaluation of relative expression levels of endogenous Jun family proteins in Th17 cells. The same amounts of FLAG–JunB, FLAG–c-Jun or FLAG–JunD, which were expressed in HEK293T cells, were estimated by immunoblot with an anti-FLAG antibody (M2) (left panel). Serially diluted proteins and the Th17 cell lysate were subjected to immunoblot analysis with anti-JunB, anti-c-Jun, or anti-JunD antibodies (middle panel), followed by quantification with Odyssey Infrared Imaging System. FLAG-tagged and endogenous proteins were denoted by white and black arrowheads, respectively. Relative protein levels of endogenous JunB, c-Jun, and JunD were shown in the right panel in (A) . ( B ) Real-time PCR analysis for relative mRNA copy numbers of Junb and Jun in naive CD4 + T cells and Th17 cells. mRNA copy numbers were estimated from standard curves that were generated using known numbers of a plasmid encoding Junb or Jun . Data are presented as mean ± SD.

Techniques Used: Expressing, Imaging, Real-time Polymerase Chain Reaction, Generated, Plasmid Preparation

57) Product Images from "Overexpression of c-Jun contributes to sorafenib resistance in human hepatoma cell lines"

Article Title: Overexpression of c-Jun contributes to sorafenib resistance in human hepatoma cell lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0174153

Sorafenib enhanced expression of osteopontin, an AP-1 target gene, in human hepatoma cell lines. (A, B) Knockdown of c-Jun decreased expression of osteopontin after 48 hours of transfection into PLC/PRF/5 cells with siRNA against c-Jun (si-c-Jun) or si-control (si-C). Lysates from transfected cells were immunoblotted with antibodies against osteopontin or β-tubulin. β-tubulin was used as internal control. (C, D) Western blot analyses of osteopontin and β-tubulin expression in PLC/PRF/5 cells treated with or without 10 μM sorafenib for 12 hours. (E, F) Western blot analyses of osteopontin and β-tubulin expression in HepG2.2.15 cells treated with or without 10 μM sorafenib for 12 hours. Densitometric analyses were performed with ImageJ software. Data are presented as mean ± SD of triplicate samples. * p
Figure Legend Snippet: Sorafenib enhanced expression of osteopontin, an AP-1 target gene, in human hepatoma cell lines. (A, B) Knockdown of c-Jun decreased expression of osteopontin after 48 hours of transfection into PLC/PRF/5 cells with siRNA against c-Jun (si-c-Jun) or si-control (si-C). Lysates from transfected cells were immunoblotted with antibodies against osteopontin or β-tubulin. β-tubulin was used as internal control. (C, D) Western blot analyses of osteopontin and β-tubulin expression in PLC/PRF/5 cells treated with or without 10 μM sorafenib for 12 hours. (E, F) Western blot analyses of osteopontin and β-tubulin expression in HepG2.2.15 cells treated with or without 10 μM sorafenib for 12 hours. Densitometric analyses were performed with ImageJ software. Data are presented as mean ± SD of triplicate samples. * p

Techniques Used: Expressing, Transfection, Planar Chromatography, Western Blot, Software

58) Product Images from "Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction"

Article Title: Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction

Journal: Scientific Reports

doi: 10.1038/srep27995

Activation of activator protein 1 (AP-1) is a critical event in PMs-induced up-regulation of COX2 expression. Cells were pre-incubated with AP-1 inhibitor (tanshinone IIA; Tan) (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. ( A) COX2 protein expression (open bars) and prostaglandin E2 (PGE2) production (shaded bars) were determined by Western blotting and ELISA as described in Fig. 1 . (B,C ) Cells were pre-incubated with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. The nuclear extract levels of c-Jun and c-Fos protein were detected using phospho-c-Jun and phospho-c-Fos antibodies, determined by Western blotting (Fig. 6 C ). Lamin-B1 was used as the loading control. ( D) Cells were pre-incubated with U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or Tan (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. Electrophoretic mobility-shift assay for assessment of AP-1 DNA binding activity, as described in “Materials and methods”. Data are expressed as mean ± standard error of the mean, based on three independent experiments. * P
Figure Legend Snippet: Activation of activator protein 1 (AP-1) is a critical event in PMs-induced up-regulation of COX2 expression. Cells were pre-incubated with AP-1 inhibitor (tanshinone IIA; Tan) (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. ( A) COX2 protein expression (open bars) and prostaglandin E2 (PGE2) production (shaded bars) were determined by Western blotting and ELISA as described in Fig. 1 . (B,C ) Cells were pre-incubated with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. The nuclear extract levels of c-Jun and c-Fos protein were detected using phospho-c-Jun and phospho-c-Fos antibodies, determined by Western blotting (Fig. 6 C ). Lamin-B1 was used as the loading control. ( D) Cells were pre-incubated with U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or Tan (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. Electrophoretic mobility-shift assay for assessment of AP-1 DNA binding activity, as described in “Materials and methods”. Data are expressed as mean ± standard error of the mean, based on three independent experiments. * P

Techniques Used: Activation Assay, Expressing, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

59) Product Images from "Icaritin Shows Potent Anti-Leukemia Activity on Chronic Myeloid Leukemia In Vitro and In Vivo by Regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT Signalings"

Article Title: Icaritin Shows Potent Anti-Leukemia Activity on Chronic Myeloid Leukemia In Vitro and In Vivo by Regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT Signalings

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023720

Effects of Icaritin on MAPK/ERK/JNK signaling pathways and Jak2/Stat3/Akt axes. A. Icaritin could up-regulate phospho-JNK, or phospho-C-Jun ( Fig.5A- a, b. ), and down-regulate phosphor-ERK, phospho-P-38 expressions ( Fig.5A-c, d ) with dose-dependent manner. While JNK, C-jun, ERK, p38 ( Fig.5A-e, f, g, h ) expressions were not influenced by Icaritin. Normalization was performed using β-actin ( Fig.5A-i ). B. Icaritin diminishes constitutive activation of JAK-2, p-Stat3 and p-Akt in K562 cells at time- or dose-dependent manner. K562 cells were treated with 8 µM Icaritin for 1-24 hours (left panel), or with different concentrations of Icaritin (0–64 µM) (right panel). After which the cells were lysed and subjected to Western blot analysis to monitor expression of JAK-2, phosphorylated Stat3 and Akt.
Figure Legend Snippet: Effects of Icaritin on MAPK/ERK/JNK signaling pathways and Jak2/Stat3/Akt axes. A. Icaritin could up-regulate phospho-JNK, or phospho-C-Jun ( Fig.5A- a, b. ), and down-regulate phosphor-ERK, phospho-P-38 expressions ( Fig.5A-c, d ) with dose-dependent manner. While JNK, C-jun, ERK, p38 ( Fig.5A-e, f, g, h ) expressions were not influenced by Icaritin. Normalization was performed using β-actin ( Fig.5A-i ). B. Icaritin diminishes constitutive activation of JAK-2, p-Stat3 and p-Akt in K562 cells at time- or dose-dependent manner. K562 cells were treated with 8 µM Icaritin for 1-24 hours (left panel), or with different concentrations of Icaritin (0–64 µM) (right panel). After which the cells were lysed and subjected to Western blot analysis to monitor expression of JAK-2, phosphorylated Stat3 and Akt.

Techniques Used: Activation Assay, Western Blot, Expressing

60) Product Images from "Transcriptional regulation of human osteopontin promoter by histone deacetylase inhibitor, trichostatin A in cervical cancer cells"

Article Title: Transcriptional regulation of human osteopontin promoter by histone deacetylase inhibitor, trichostatin A in cervical cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-178

PMA induces AP-1 DNA binding to the OPN promoter in HeLa cells . A . HeLa cells were treated with PMA (50 ng/ml) for 2 h. Nuclear extracts were prepared and incubated with 32 P-labeled probe containing AP-1 binding site of OPN promoter and analyzed by EMSA. B . For supershift assay, nuclear extracts from PMA-treated HeLa cells were incubated with anti-c-Jun or anti-c-Fos antibody and then analyzed by EMSA. C . HeLa cells were pretreated with TSA (0.5 μM) for 1 h and then with PMA (50 ng/ml) for 2 h. Cross-linked chromatin fragments were immunoprecipitated with anti-p-c-Jun antibody and was PCR amplified using specific primers derived from the region of OPN promoter containing AP-1 binding site. For negative controls, normal mouse IgG was used or specific antibody was omitted.
Figure Legend Snippet: PMA induces AP-1 DNA binding to the OPN promoter in HeLa cells . A . HeLa cells were treated with PMA (50 ng/ml) for 2 h. Nuclear extracts were prepared and incubated with 32 P-labeled probe containing AP-1 binding site of OPN promoter and analyzed by EMSA. B . For supershift assay, nuclear extracts from PMA-treated HeLa cells were incubated with anti-c-Jun or anti-c-Fos antibody and then analyzed by EMSA. C . HeLa cells were pretreated with TSA (0.5 μM) for 1 h and then with PMA (50 ng/ml) for 2 h. Cross-linked chromatin fragments were immunoprecipitated with anti-p-c-Jun antibody and was PCR amplified using specific primers derived from the region of OPN promoter containing AP-1 binding site. For negative controls, normal mouse IgG was used or specific antibody was omitted.

Techniques Used: Binding Assay, Incubation, Labeling, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Derivative Assay

TSA blocks PMA-induced c-Jun but not c-Fos expression in HeLa cells . A and C . HeLa cells were pretreated with TSA (0-1 μM) for 1 h followed by treatment with PMA (50 ng/ml) for 2 h. Cell lysates (50 μg) containing equal amount of total proteins were analyzed by western blot using either anti-c-Jun or anti-c-Fos antibody. B and D . HeLa cells were pretreated with TSA and then with PMA under similar conditions as described above. Total RNA was isolated and the levels of c-jun and c-fos mRNAs were detected by semiquantitative RT-PCR. Actin was used as control.
Figure Legend Snippet: TSA blocks PMA-induced c-Jun but not c-Fos expression in HeLa cells . A and C . HeLa cells were pretreated with TSA (0-1 μM) for 1 h followed by treatment with PMA (50 ng/ml) for 2 h. Cell lysates (50 μg) containing equal amount of total proteins were analyzed by western blot using either anti-c-Jun or anti-c-Fos antibody. B and D . HeLa cells were pretreated with TSA and then with PMA under similar conditions as described above. Total RNA was isolated and the levels of c-jun and c-fos mRNAs were detected by semiquantitative RT-PCR. Actin was used as control.

Techniques Used: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

61) Product Images from "Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability"

Article Title: Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-8-27

The degradation of c-Jun is accelerated in CSN knockdown cells . (a) CHX chase experiments were performed with siGFP, siCSN1, siCSN3 and siCSN5 cells. After indicated time aliquots of cell lysates were analyzed by Western blotting using the anti-c-Jun antibody. The same samples were probed with the antibody against the 26S proteasome base subunit RPN2/S1 as a loading control. (b) CSN5 knockdown cells were transfected with CSN5wt as in Fig. 1d or with CSN5D151N and 24 h after transfection CHX chase experiments were carried out as in (a). The middle panel shows Western blots with the anti-Flag antibody visualizing the expressed CSN5wt or CSN5D151N proteins (c) CSN5wt was overexpressed in siCSN1 cells and after 24 h CHX experiments were performed as in (a). In all CHX experiments a band just above c-Jun appeared after 20 – 30 min, which cross-reacted with the anti-c-Jun antibody (in some blots it was cut off). This protein might be a modified c-Jun. The nature of this modification is currently unknown. Both the putative modified c-Jun as well as unmodified c-Jun disappeared during the experiment.
Figure Legend Snippet: The degradation of c-Jun is accelerated in CSN knockdown cells . (a) CHX chase experiments were performed with siGFP, siCSN1, siCSN3 and siCSN5 cells. After indicated time aliquots of cell lysates were analyzed by Western blotting using the anti-c-Jun antibody. The same samples were probed with the antibody against the 26S proteasome base subunit RPN2/S1 as a loading control. (b) CSN5 knockdown cells were transfected with CSN5wt as in Fig. 1d or with CSN5D151N and 24 h after transfection CHX chase experiments were carried out as in (a). The middle panel shows Western blots with the anti-Flag antibody visualizing the expressed CSN5wt or CSN5D151N proteins (c) CSN5wt was overexpressed in siCSN1 cells and after 24 h CHX experiments were performed as in (a). In all CHX experiments a band just above c-Jun appeared after 20 – 30 min, which cross-reacted with the anti-c-Jun antibody (in some blots it was cut off). This protein might be a modified c-Jun. The nature of this modification is currently unknown. Both the putative modified c-Jun as well as unmodified c-Jun disappeared during the experiment.

Techniques Used: Western Blot, Transfection, Modification

62) Product Images from "Bile acids and cytokines inhibit the human cholesterol 7?-hydroxylase gene via the JNK/c-Jun pathway"

Article Title: Bile acids and cytokines inhibit the human cholesterol 7?-hydroxylase gene via the JNK/c-Jun pathway

Journal: Hepatology (Baltimore, Md.)

doi: 10.1002/hep.21183

Chromatin immunoprecipitation assay of HNF4α, PGC-1α, and c-Jun binding to the CYP7A1 chromatin. A. Anti-HNF4α antibody was used to precipitate chromatin from HepG2 cells treated with IL-1β (10 ng/ml) or CDCA (50 μM). B. Anti-c-Jun antibody was used to immunoprecipitate chromatin. C. HepG2 cells were transfected with cJun or JNK1 expression plasmid or pcDNA3 empty vector, and anti-HNF4α antibody was used to immunoprecipitate chromatin. D. HepG2 cells were transfected with HA-PGC-1α, and co-transfected with a c-Jun expression plasmid or pcDNA3 empty vector. A 391 bp fragment containing the BAREI and BAREII regions of the CYP7A1 promoter was PCR amplified and analyzed on a 1.5% agarose gel.
Figure Legend Snippet: Chromatin immunoprecipitation assay of HNF4α, PGC-1α, and c-Jun binding to the CYP7A1 chromatin. A. Anti-HNF4α antibody was used to precipitate chromatin from HepG2 cells treated with IL-1β (10 ng/ml) or CDCA (50 μM). B. Anti-c-Jun antibody was used to immunoprecipitate chromatin. C. HepG2 cells were transfected with cJun or JNK1 expression plasmid or pcDNA3 empty vector, and anti-HNF4α antibody was used to immunoprecipitate chromatin. D. HepG2 cells were transfected with HA-PGC-1α, and co-transfected with a c-Jun expression plasmid or pcDNA3 empty vector. A 391 bp fragment containing the BAREI and BAREII regions of the CYP7A1 promoter was PCR amplified and analyzed on a 1.5% agarose gel.

Techniques Used: Chromatin Immunoprecipitation, Pyrolysis Gas Chromatography, Binding Assay, Transfection, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Co-immunoprecipitation assay of HNF4α and c-Jun interaction-HepG2 cell extracts treated with IL-1β (5 ng/ml) for the time indicated were immunoprecipitated with rabbit anti-HNF4α antibody as described in Materials and Methods. Immunobolot analysis was performed with goat anti-HNF4α, anti-c-Jun and anti-phospho c-Jun antibodies. Five percent of the cell lysate was immunoblotted with anti-actin antibody as internal control. Rabbit non-immune IgG was used as a negative control. The ratios of c-Jun to HNF4α and phospho-c-Jun to HNF4α are indicated below the panels.
Figure Legend Snippet: Co-immunoprecipitation assay of HNF4α and c-Jun interaction-HepG2 cell extracts treated with IL-1β (5 ng/ml) for the time indicated were immunoprecipitated with rabbit anti-HNF4α antibody as described in Materials and Methods. Immunobolot analysis was performed with goat anti-HNF4α, anti-c-Jun and anti-phospho c-Jun antibodies. Five percent of the cell lysate was immunoblotted with anti-actin antibody as internal control. Rabbit non-immune IgG was used as a negative control. The ratios of c-Jun to HNF4α and phospho-c-Jun to HNF4α are indicated below the panels.

Techniques Used: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control

63) Product Images from "TGF-β3 Promotes MUC5AC Hyper-Expression by Modulating Autophagy Pathway in Airway Epithelium"

Article Title: TGF-β3 Promotes MUC5AC Hyper-Expression by Modulating Autophagy Pathway in Airway Epithelium

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.06.032

TGF-β3-induced autophagy contributed to increased MUC5AC production by activating the AP1. (a-b) 16HBE cells were transduced with ATG5-siRNA lentivirus (a) and BECN1-siRNA lentivirus (b), respectively. After treating the cells with TGF-β3 (10 ng/ml) for 24 h, phospho-c-Jun and c-Jun were detected by western blot. (c-d) 16HBE cells were treated with TGF-β3 in the presence of 3-MA (c) or Baf A1 (d). Then, the expression of phospho-c-Jun and c-Jun were detected using western blot assay. (e) 16HBE cells were transfected with Smad2/3-siRNA. After treating the cells with TGF-β3 (10 ng/ml) for 24 h, phospho-c-Jun and c-Jun were detected by western blot. (f) 16HBE cells were transfected with c-Jun-siRNA, after treating with TGF-β3 (10 ng/ml) for 24 h, and then phospho-c-Jun and c-Jun were detected by western blot. (g) Representative immunofluorescence images of TGF-β3-induced MUC5AC in 16HBE cells were transfected with c-Jun-siRNA. (h) Quantitation of fluorescence intensity of MUC5AC (each group n = 10 images for quantification). (i) 16HBE cells were transfected with c-Jun-siRNA. Real-time PCR was performed to detect the expression of MUC5AC gene after treated with TGF-β3 (10 ng/ml). Data are representative of the three independent experiments and are presented as means ± s.d . *P
Figure Legend Snippet: TGF-β3-induced autophagy contributed to increased MUC5AC production by activating the AP1. (a-b) 16HBE cells were transduced with ATG5-siRNA lentivirus (a) and BECN1-siRNA lentivirus (b), respectively. After treating the cells with TGF-β3 (10 ng/ml) for 24 h, phospho-c-Jun and c-Jun were detected by western blot. (c-d) 16HBE cells were treated with TGF-β3 in the presence of 3-MA (c) or Baf A1 (d). Then, the expression of phospho-c-Jun and c-Jun were detected using western blot assay. (e) 16HBE cells were transfected with Smad2/3-siRNA. After treating the cells with TGF-β3 (10 ng/ml) for 24 h, phospho-c-Jun and c-Jun were detected by western blot. (f) 16HBE cells were transfected with c-Jun-siRNA, after treating with TGF-β3 (10 ng/ml) for 24 h, and then phospho-c-Jun and c-Jun were detected by western blot. (g) Representative immunofluorescence images of TGF-β3-induced MUC5AC in 16HBE cells were transfected with c-Jun-siRNA. (h) Quantitation of fluorescence intensity of MUC5AC (each group n = 10 images for quantification). (i) 16HBE cells were transfected with c-Jun-siRNA. Real-time PCR was performed to detect the expression of MUC5AC gene after treated with TGF-β3 (10 ng/ml). Data are representative of the three independent experiments and are presented as means ± s.d . *P

Techniques Used: Transduction, Western Blot, Expressing, Transfection, Immunofluorescence, Quantitation Assay, Fluorescence, Real-time Polymerase Chain Reaction

Smad2/3 pathway is involved in TGF-β3 induced autophagy and MUC5AC. (a) 16HBE cells were transfected with Smad2/3-siRNA. After treating the cells with TGF-β3 (10 ng/ml) for 24 h, LC3B, BECN1, ATG5, phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by western blot. (b) Relative changes in the density of LC3B II were detected. (c) 16HBE cells that stably expressed mCherry-EGFP-LC3 fusion protein were transfected with Smad2/3-siRNA. After treating with TGF-β3 (10 ng/ml) for 24 h, autophagosomes were observed under confocal microscope (2000× magnification) in 16HBE cells. Bar scale, 5 mm. (d) Quantification of the number of LC3 puncta (each group n = 10 images for quantification). (e) 16HBE cells were transfected with Smad2/3-siRNA. Real-time PCR was performed to detect the expression of MUC5AC gene after treated with TGF-β3 (10 ng/ml). (f) Representative immunofluorescence images of TGF-β3-induced MUC5AC in 16HBE cells transfected with Smad2/3-siRNA. (g) Quantitation of the fluorescence intensity of MUC5AC (each group n = 10 images for quantification). Data are representative of three independent experiments and are presented as means ± s.d . *P
Figure Legend Snippet: Smad2/3 pathway is involved in TGF-β3 induced autophagy and MUC5AC. (a) 16HBE cells were transfected with Smad2/3-siRNA. After treating the cells with TGF-β3 (10 ng/ml) for 24 h, LC3B, BECN1, ATG5, phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by western blot. (b) Relative changes in the density of LC3B II were detected. (c) 16HBE cells that stably expressed mCherry-EGFP-LC3 fusion protein were transfected with Smad2/3-siRNA. After treating with TGF-β3 (10 ng/ml) for 24 h, autophagosomes were observed under confocal microscope (2000× magnification) in 16HBE cells. Bar scale, 5 mm. (d) Quantification of the number of LC3 puncta (each group n = 10 images for quantification). (e) 16HBE cells were transfected with Smad2/3-siRNA. Real-time PCR was performed to detect the expression of MUC5AC gene after treated with TGF-β3 (10 ng/ml). (f) Representative immunofluorescence images of TGF-β3-induced MUC5AC in 16HBE cells transfected with Smad2/3-siRNA. (g) Quantitation of the fluorescence intensity of MUC5AC (each group n = 10 images for quantification). Data are representative of three independent experiments and are presented as means ± s.d . *P

Techniques Used: Transfection, Western Blot, Stable Transfection, Microscopy, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Quantitation Assay, Fluorescence

64) Product Images from "NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes"

Article Title: NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-10-35

AP-1 (c-Fos/c-Jun) is essential for BK-induced MMP-9 expression through a Ca 2+ /PKC-α/Nox2/ROS cascade. ( A ) Cells were pretreated without or with tanshinone IIA (TSIIA, 10 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated without or with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h before exposure to 10 nM BK for 30 min. The nuclear fraction was collected and analyzed by Western blotting using an anti-c-Fos, phospho-c-Jun, or c-Jun antibody. ( C ) Cells were transiently cotransfected with pAP1-Luc and pGal for 24 h, pretreated with BAPTA (BAP), TG, Gö, Apo, DPI, or NAC for 1 h and then incubated with BK for 6 h. The AP-1 promoter activity in the cell lysates was determined. ( D ) Cells were transfected with scramble (scra) or c-Fos/c-Jun siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. Data are expressed as the mean ± SEM (n = 3). * P
Figure Legend Snippet: AP-1 (c-Fos/c-Jun) is essential for BK-induced MMP-9 expression through a Ca 2+ /PKC-α/Nox2/ROS cascade. ( A ) Cells were pretreated without or with tanshinone IIA (TSIIA, 10 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated without or with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h before exposure to 10 nM BK for 30 min. The nuclear fraction was collected and analyzed by Western blotting using an anti-c-Fos, phospho-c-Jun, or c-Jun antibody. ( C ) Cells were transiently cotransfected with pAP1-Luc and pGal for 24 h, pretreated with BAPTA (BAP), TG, Gö, Apo, DPI, or NAC for 1 h and then incubated with BK for 6 h. The AP-1 promoter activity in the cell lysates was determined. ( D ) Cells were transfected with scramble (scra) or c-Fos/c-Jun siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. Data are expressed as the mean ± SEM (n = 3). * P

Techniques Used: Expressing, Western Blot, Incubation, Activity Assay, Transfection, Zymography

65) Product Images from "Involvement of the DNA Repair Protein hHR23 in p53 Degradation"

Article Title: Involvement of the DNA Repair Protein hHR23 in p53 Degradation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.24.8960-8969.2003

Downregulation of hHR23 results in accumulation of endogenous p53. (A) U2OS cells (which express endogenous wt p53) were transfected with an expression construct for HA-tagged hHR23 or with an expression construct for CD44 for control. Twenty-four hours h after transfection, levels of p53, c-Jun, hHR23, or CD44 were determined by immunofluorescence using the anti-p53 antibody CM-1, the anti-c-Jun antibody sc-45, a monoclonal anti-HA antibody, and a monoclonal antibody against CD44. p53 and c-Jun are stained in red. hHR23 and CD44 are stained in green. (B) Primary human fibroblasts were microinjected with an expression vector encoding GFP either alone, in combination with control siRNA, or in combination with siRNAs directed against mRNAs encoding hHR23A and hHR23B. Three days after microinjection, p53 expression was determined by immunofluorescence using the p53-specific antibody DO-1. GFP expression is shown in green, and the p53 protein is stained in red.
Figure Legend Snippet: Downregulation of hHR23 results in accumulation of endogenous p53. (A) U2OS cells (which express endogenous wt p53) were transfected with an expression construct for HA-tagged hHR23 or with an expression construct for CD44 for control. Twenty-four hours h after transfection, levels of p53, c-Jun, hHR23, or CD44 were determined by immunofluorescence using the anti-p53 antibody CM-1, the anti-c-Jun antibody sc-45, a monoclonal anti-HA antibody, and a monoclonal antibody against CD44. p53 and c-Jun are stained in red. hHR23 and CD44 are stained in green. (B) Primary human fibroblasts were microinjected with an expression vector encoding GFP either alone, in combination with control siRNA, or in combination with siRNAs directed against mRNAs encoding hHR23A and hHR23B. Three days after microinjection, p53 expression was determined by immunofluorescence using the p53-specific antibody DO-1. GFP expression is shown in green, and the p53 protein is stained in red.

Techniques Used: Transfection, Expressing, Construct, Immunofluorescence, Staining, Plasmid Preparation

66) Product Images from "Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1"

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.14.5933-5946.2005

Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P
Figure Legend Snippet: Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P

Techniques Used: Western Blot

67) Product Images from "Laminin-\u03b11 LG4\u20135 domain binding to dystroglycan mediates muscle cell survival, growth, and the AP-1 and NF-\u03baB transcription factors but also has adverse effects"

Article Title: Laminin-\u03b11 LG4\u20135 domain binding to dystroglycan mediates muscle cell survival, growth, and the AP-1 and NF-\u03baB transcription factors but also has adverse effects

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00118.2011

1E3 and laminin induce activating protein-1 (AP-1) element DNA-binding activity, and the binding complex was blocked and shifted by a specific phosphorylated c-jun antibody.  A : C2C12 myotubes were stimulated with laminin (3 μg/ml) for an hour
Figure Legend Snippet: 1E3 and laminin induce activating protein-1 (AP-1) element DNA-binding activity, and the binding complex was blocked and shifted by a specific phosphorylated c-jun antibody. A : C2C12 myotubes were stimulated with laminin (3 μg/ml) for an hour

Techniques Used: Binding Assay, Activity Assay

68) Product Images from "Rosuvastatin suppresses platelet-derived growth factor-BB-induced vascular smooth muscle cell proliferation and migration via the MAPK signaling pathway"

Article Title: Rosuvastatin suppresses platelet-derived growth factor-BB-induced vascular smooth muscle cell proliferation and migration via the MAPK signaling pathway

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2013.1265

Rosuvastatin inhibited the mitogen-activated protein kinase (MAPK) signaling pathway activated by platelet-derived growth factor-BB (PDGF-BB) in vascular smooth muscle cells (VSMCs). Con, VSMCs were cultured without any treatment; NC, VSMCs were treated only with PDGF-BB (20 ng/ml) for 48 h; rosuvastatin, VSMCs were treated with rosuvastatin (10 μ M) and PDGF-BB (20 ng/ml) for 48 h. Western blot analysis was used to determine the protein expression of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), ERK, phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK) and JNK. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
Figure Legend Snippet: Rosuvastatin inhibited the mitogen-activated protein kinase (MAPK) signaling pathway activated by platelet-derived growth factor-BB (PDGF-BB) in vascular smooth muscle cells (VSMCs). Con, VSMCs were cultured without any treatment; NC, VSMCs were treated only with PDGF-BB (20 ng/ml) for 48 h; rosuvastatin, VSMCs were treated with rosuvastatin (10 μ M) and PDGF-BB (20 ng/ml) for 48 h. Western blot analysis was used to determine the protein expression of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), ERK, phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK) and JNK. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference.

Techniques Used: Derivative Assay, Cell Culture, Western Blot, Expressing

69) Product Images from "c-Jun Is Required for Nuclear Factor-κB–Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages"

Article Title: c-Jun Is Required for Nuclear Factor-κB–Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2016-0028OC

Extracellular signal-regulated protein kinase 1/2 inhibition blocks LPS-stimulated NF-κB phosphorylation. MH-S cells were incubated with U0126 for 30 minutes and then treated with LPS for 0–90 minutes. Total lysates were isolated, blotted onto membrane, and probed with phospho-c-Jun (serine 65) ( A ) or phospho-NF-κB (pNF-κB) (p65, serine 536) ( B ) antibodies. Membranes were stripped and probed with β-actin antibody to demonstrate equal protein loading. Relative band intensities from a representative blot ( n = 2) are shown.
Figure Legend Snippet: Extracellular signal-regulated protein kinase 1/2 inhibition blocks LPS-stimulated NF-κB phosphorylation. MH-S cells were incubated with U0126 for 30 minutes and then treated with LPS for 0–90 minutes. Total lysates were isolated, blotted onto membrane, and probed with phospho-c-Jun (serine 65) ( A ) or phospho-NF-κB (pNF-κB) (p65, serine 536) ( B ) antibodies. Membranes were stripped and probed with β-actin antibody to demonstrate equal protein loading. Relative band intensities from a representative blot ( n = 2) are shown.

Techniques Used: Inhibition, Incubation, Isolation

70) Product Images from "Characterization of Short Range DNA Looping in Endotoxin-mediated Transcription of the Murine Inducible Nitric-oxide Synthase (iNOS) Gene *"

Article Title: Characterization of Short Range DNA Looping in Endotoxin-mediated Transcription of the Murine Inducible Nitric-oxide Synthase (iNOS) Gene *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M804062200

a , immunoprecipitation ( IP ) of p300, NF-κB, and AP-1 complex. Nuclear protein from ANA-1 cells was incubated with anti-p65 Ab or anti-c-Jun Ab (R D Systems), and protein G-agarose, loaded onto 4–20% SDS-PAGE gel, and transfered
Figure Legend Snippet: a , immunoprecipitation ( IP ) of p300, NF-κB, and AP-1 complex. Nuclear protein from ANA-1 cells was incubated with anti-p65 Ab or anti-c-Jun Ab (R D Systems), and protein G-agarose, loaded onto 4–20% SDS-PAGE gel, and transfered

Techniques Used: Immunoprecipitation, Incubation, SDS Page

71) Product Images from "The Role of Cyclooxygenase-2, Interleukin-1β and Fibroblast Growth Factor -2 in the Activation of Matrix Metalloproteinase-1 in Sheared-Chondrocytes and Articular Cartilage"

Article Title: The Role of Cyclooxygenase-2, Interleukin-1β and Fibroblast Growth Factor -2 in the Activation of Matrix Metalloproteinase-1 in Sheared-Chondrocytes and Articular Cartilage

Journal: Scientific Reports

doi: 10.1038/srep10412

NF-κB and c-Jun are key transcriptional factors involved in the synthesis of MMP-1 in sheared human T/C-28a2 chondrocytes. In promoter assay experiments, T/C-28a2 cells were transiently transfected with a series of truncated or mutated pMMP-1-luc plasmid ( a , b ). The luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit, which were normalized to the Renilla luciferase activities ( a , b ). In select experiments, nuclear extracts were isolated, and c-Jun- and NF-κB-specific DNA-protein complex formation was determined by EMSA ( c , d ). In separate experiments, cross-linked chromatin was immunoprecipitated using an anti-c-Jun ( e ) or anti-p65 antibody ( f ). In the ChIP assays, the anti-RNA polymerase II antibody was used as a positive control. DNA purified from immunoprecipitated ( IP ) and preimmune ( Input ) specimens was subjected to qPCR amplification using primers for the mmp-1 promoter. All experiments are representative of three independent experiments, all revealing similar results ( e , f ). The data represent the means ± S.E. of three independent experiments. *, p
Figure Legend Snippet: NF-κB and c-Jun are key transcriptional factors involved in the synthesis of MMP-1 in sheared human T/C-28a2 chondrocytes. In promoter assay experiments, T/C-28a2 cells were transiently transfected with a series of truncated or mutated pMMP-1-luc plasmid ( a , b ). The luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit, which were normalized to the Renilla luciferase activities ( a , b ). In select experiments, nuclear extracts were isolated, and c-Jun- and NF-κB-specific DNA-protein complex formation was determined by EMSA ( c , d ). In separate experiments, cross-linked chromatin was immunoprecipitated using an anti-c-Jun ( e ) or anti-p65 antibody ( f ). In the ChIP assays, the anti-RNA polymerase II antibody was used as a positive control. DNA purified from immunoprecipitated ( IP ) and preimmune ( Input ) specimens was subjected to qPCR amplification using primers for the mmp-1 promoter. All experiments are representative of three independent experiments, all revealing similar results ( e , f ). The data represent the means ± S.E. of three independent experiments. *, p

Techniques Used: Promoter Assay, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Isolation, Immunoprecipitation, Chromatin Immunoprecipitation, Positive Control, Purification, Real-time Polymerase Chain Reaction, Amplification

72) Product Images from "c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription"

Article Title: c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.1.12-22.2002

Association between c-Jun and HIF-1α. (A) COS-7 cells were cotransfected with 8 μg of pcDNA3α1 and pRSV c-Jun (left) or pCMV TAM67 (right) expression vector. After 40 h, the cells were incubated in normoxia or hypoxia (Hx) for 4 h, and nuclear extracts were obtained. These extracts were immunoprecipitated (IP) with polyclonal antibodies against the DNA binding domain (c-Jun and c-Jun 1) or the transactivation domain (c-Jun 2) of c-Jun or a control antibody (C) and immunoblotted with anti-HIF-1α (top) and anti-c-Jun (bottom) antibodies; as a control for protein expression, aliquots of the lysates (representing 1/10 of each immunoprecipitation reaction) (−) were also subjected to Western blotting with antibodies against HIF-1α and c-Jun. (B) HIF-1α (α1), c-Jun, HIF-1β (β), and ATF-2 were in vitro translated or cotranslated in the presence of [ 35 S]methionine and immunoprecipitated with antibodies against HIF-1α (α1), c-Jun, or ATF-2. The immunocomplexes were analyzed by SDS-PAGE (right); the autoradiograph on the left shows 1/10 of each translation reaction as a control. +, present; −, absent. Molecular weight markers are shown on the right.
Figure Legend Snippet: Association between c-Jun and HIF-1α. (A) COS-7 cells were cotransfected with 8 μg of pcDNA3α1 and pRSV c-Jun (left) or pCMV TAM67 (right) expression vector. After 40 h, the cells were incubated in normoxia or hypoxia (Hx) for 4 h, and nuclear extracts were obtained. These extracts were immunoprecipitated (IP) with polyclonal antibodies against the DNA binding domain (c-Jun and c-Jun 1) or the transactivation domain (c-Jun 2) of c-Jun or a control antibody (C) and immunoblotted with anti-HIF-1α (top) and anti-c-Jun (bottom) antibodies; as a control for protein expression, aliquots of the lysates (representing 1/10 of each immunoprecipitation reaction) (−) were also subjected to Western blotting with antibodies against HIF-1α and c-Jun. (B) HIF-1α (α1), c-Jun, HIF-1β (β), and ATF-2 were in vitro translated or cotranslated in the presence of [ 35 S]methionine and immunoprecipitated with antibodies against HIF-1α (α1), c-Jun, or ATF-2. The immunocomplexes were analyzed by SDS-PAGE (right); the autoradiograph on the left shows 1/10 of each translation reaction as a control. +, present; −, absent. Molecular weight markers are shown on the right.

Techniques Used: Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Binding Assay, Western Blot, In Vitro, SDS Page, Autoradiography, Molecular Weight

c-Jun binding to VEGF 5′ UTR HIF-1 consensus sequence in endothelial cells. (A) Electrophoretic mobility shift assay of nuclear extracts obtained from primary endothelial cells either untreated or subjected to hypoxia (Hx) for 4 h; these extracts were incubated (3 μg per lane) with a 32 P-labeled probe which contains the HIF-1 DNA binding consensus sequence of the VEGF 5′ UTR (HIF-1; 0.5 ng per lane), showing a hypoxia-inducible DNA-protein complex (arrows). In some cases, nuclear extracts were incubated with specific antibodies (Ab) against the HIF-1α subunit (α1) (left) or c-Jun or c-Fos (middle) before the probe was added. The autoradiographs show supershifted complexes with anti-HIF-1α (asterisk) and c-Jun (arrowhead) antibodies, whereas anti-c-Fos had no effect; (right) electrophoretic mobility shift assay performed as described above but with a 32 P-labeled probe containing an AP-1 consensus sequence from the CD11c promoter (AP-1) used as a positive control. Supershifted complexes can be observed with both anti-c-Jun (open arrow) and anti c-Fos (open arrowhead) antibodies. (B) In vitro-translated (Ret.) pcDNA3 (C), c-Jun, or HIF-1 (α1 plus β subunits) was incubated with the same labeled probes as for panel A (1 ng per lane). Specific complexes with AP-1 (asterisk) and HIF-1 (arrow) probes could be observed, which could be identified, respectively, as c-Jun and the HIF-1 α subunit (arrowhead) with specific antibodies (Ab) against these factors. +, present; −, absent.
Figure Legend Snippet: c-Jun binding to VEGF 5′ UTR HIF-1 consensus sequence in endothelial cells. (A) Electrophoretic mobility shift assay of nuclear extracts obtained from primary endothelial cells either untreated or subjected to hypoxia (Hx) for 4 h; these extracts were incubated (3 μg per lane) with a 32 P-labeled probe which contains the HIF-1 DNA binding consensus sequence of the VEGF 5′ UTR (HIF-1; 0.5 ng per lane), showing a hypoxia-inducible DNA-protein complex (arrows). In some cases, nuclear extracts were incubated with specific antibodies (Ab) against the HIF-1α subunit (α1) (left) or c-Jun or c-Fos (middle) before the probe was added. The autoradiographs show supershifted complexes with anti-HIF-1α (asterisk) and c-Jun (arrowhead) antibodies, whereas anti-c-Fos had no effect; (right) electrophoretic mobility shift assay performed as described above but with a 32 P-labeled probe containing an AP-1 consensus sequence from the CD11c promoter (AP-1) used as a positive control. Supershifted complexes can be observed with both anti-c-Jun (open arrow) and anti c-Fos (open arrowhead) antibodies. (B) In vitro-translated (Ret.) pcDNA3 (C), c-Jun, or HIF-1 (α1 plus β subunits) was incubated with the same labeled probes as for panel A (1 ng per lane). Specific complexes with AP-1 (asterisk) and HIF-1 (arrow) probes could be observed, which could be identified, respectively, as c-Jun and the HIF-1 α subunit (arrowhead) with specific antibodies (Ab) against these factors. +, present; −, absent.

Techniques Used: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Positive Control, In Vitro

Effect of c-Jun or its dominant-negative mutant on HIF-1α expression. (A) COS-7 cells were transfected with control empty vector (MOCK) or increasing amounts of c-Jun expression vector (c-Jun). After transfection, the cells were grown under normoxic (N) or hypoxic (Hx) conditions for 5 h. Nuclear extracts were obtained and subjected to immunoblotting (20 μg per lane) with an anti-HIF-1α antibody (top) or an anti-c-Jun antibody (bottom). (B) COS-7 cells were transfected with 8 μg of control empty vector (MOCK) or pCMV TAM67 (TAM67) and processed as for panel A. Molecular weight markers are shown on the left.
Figure Legend Snippet: Effect of c-Jun or its dominant-negative mutant on HIF-1α expression. (A) COS-7 cells were transfected with control empty vector (MOCK) or increasing amounts of c-Jun expression vector (c-Jun). After transfection, the cells were grown under normoxic (N) or hypoxic (Hx) conditions for 5 h. Nuclear extracts were obtained and subjected to immunoblotting (20 μg per lane) with an anti-HIF-1α antibody (top) or an anti-c-Jun antibody (bottom). (B) COS-7 cells were transfected with 8 μg of control empty vector (MOCK) or pCMV TAM67 (TAM67) and processed as for panel A. Molecular weight markers are shown on the left.

Techniques Used: Dominant Negative Mutation, Expressing, Transfection, Plasmid Preparation, Molecular Weight

Involvement of JNK pathway in functional cooperation between c-Jun and HIF-1. (A) Western blot analysis of phosphorylated c-Jun in endothelial cells under hypoxic conditions. (Top) Nuclear extracts were obtained from HUVEC grown in 1% O 2 (Hx) for 30 min (30′) and 1, 2.5, 5, and 15 h or under normoxic conditions (C) and subjected to immunoblotting with an antibody which specifically recognizes c-Jun phosphorylated at Ser 63 . (Bottom) The same membrane was stripped and reblotted with an anti-c-Jun antibody to show equivalent amounts of protein in each lane. (B) (Top) F9 cells were transfected with 0.5 μg of p9HIF1Luc luciferase reporter plasmid, together with 1 μg of pRSVc-Jun (WT), pRSVc-Jun S63A-S73A (S63/73A), or pUCRSV control vector (C); after transfection, the cells were grown under hypoxic conditions (Hx) for 16 h, and luciferase activity was determined. Data from three separate experiments are shown, normalized to control in hypoxia (set as 1); the bars represent the means of the different values for each experimental condition. ∗, P
Figure Legend Snippet: Involvement of JNK pathway in functional cooperation between c-Jun and HIF-1. (A) Western blot analysis of phosphorylated c-Jun in endothelial cells under hypoxic conditions. (Top) Nuclear extracts were obtained from HUVEC grown in 1% O 2 (Hx) for 30 min (30′) and 1, 2.5, 5, and 15 h or under normoxic conditions (C) and subjected to immunoblotting with an antibody which specifically recognizes c-Jun phosphorylated at Ser 63 . (Bottom) The same membrane was stripped and reblotted with an anti-c-Jun antibody to show equivalent amounts of protein in each lane. (B) (Top) F9 cells were transfected with 0.5 μg of p9HIF1Luc luciferase reporter plasmid, together with 1 μg of pRSVc-Jun (WT), pRSVc-Jun S63A-S73A (S63/73A), or pUCRSV control vector (C); after transfection, the cells were grown under hypoxic conditions (Hx) for 16 h, and luciferase activity was determined. Data from three separate experiments are shown, normalized to control in hypoxia (set as 1); the bars represent the means of the different values for each experimental condition. ∗, P

Techniques Used: Functional Assay, Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay

73) Product Images from "Chronic Cadmium Exposure Stimulates SDF-1 Expression in an ER? Dependent Manner"

Article Title: Chronic Cadmium Exposure Stimulates SDF-1 Expression in an ER? Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072639

ERα, c-fos and c-jun are recruited to SDF-1 promoter. ( A ) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. ( B ) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P
Figure Legend Snippet: ERα, c-fos and c-jun are recruited to SDF-1 promoter. ( A ) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. ( B ) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Prolonged exposure to cadmium enhances the interactions of ERα with c-jun and c-fos. ( A ) MCF7, Cd7 and Cd12 cell lysates were immunoprecipitated with either α-ERα or normal rabbit IgG. Proteins interacting with ERα were analyzed with Western blot analysis. ( B ) Reverse co-IP was performed with α-c-jun, α-c-fos, or normal rabbit IgG. ( C ) MCF7, Cd7 and Cd12 were plated in 6-well plates and transfected with siRNA targeting either c-jun (Ji), c-fos (Fi) or a scramble siRNA control (Ci) and collected 48 hours later for protein analysis.
Figure Legend Snippet: Prolonged exposure to cadmium enhances the interactions of ERα with c-jun and c-fos. ( A ) MCF7, Cd7 and Cd12 cell lysates were immunoprecipitated with either α-ERα or normal rabbit IgG. Proteins interacting with ERα were analyzed with Western blot analysis. ( B ) Reverse co-IP was performed with α-c-jun, α-c-fos, or normal rabbit IgG. ( C ) MCF7, Cd7 and Cd12 were plated in 6-well plates and transfected with siRNA targeting either c-jun (Ji), c-fos (Fi) or a scramble siRNA control (Ci) and collected 48 hours later for protein analysis.

Techniques Used: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Transfection

74) Product Images from "Chronic Cadmium Exposure Stimulates SDF-1 Expression in an ER? Dependent Manner"

Article Title: Chronic Cadmium Exposure Stimulates SDF-1 Expression in an ER? Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072639

ERα, c-fos and c-jun are recruited to SDF-1 promoter. ( A ) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. ( B ) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P
Figure Legend Snippet: ERα, c-fos and c-jun are recruited to SDF-1 promoter. ( A ) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. ( B ) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Prolonged exposure to cadmium enhances the interactions of ERα with c-jun and c-fos. ( A ) MCF7, Cd7 and Cd12 cell lysates were immunoprecipitated with either α-ERα or normal rabbit IgG. Proteins interacting with ERα were analyzed with Western blot analysis. ( B ) Reverse co-IP was performed with α-c-jun, α-c-fos, or normal rabbit IgG. ( C ) MCF7, Cd7 and Cd12 were plated in 6-well plates and transfected with siRNA targeting either c-jun (Ji), c-fos (Fi) or a scramble siRNA control (Ci) and collected 48 hours later for protein analysis.
Figure Legend Snippet: Prolonged exposure to cadmium enhances the interactions of ERα with c-jun and c-fos. ( A ) MCF7, Cd7 and Cd12 cell lysates were immunoprecipitated with either α-ERα or normal rabbit IgG. Proteins interacting with ERα were analyzed with Western blot analysis. ( B ) Reverse co-IP was performed with α-c-jun, α-c-fos, or normal rabbit IgG. ( C ) MCF7, Cd7 and Cd12 were plated in 6-well plates and transfected with siRNA targeting either c-jun (Ji), c-fos (Fi) or a scramble siRNA control (Ci) and collected 48 hours later for protein analysis.

Techniques Used: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Transfection

75) Product Images from "Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice"

Article Title: Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice

Journal: Clinical Interventions in Aging

doi: 10.2147/CIA.S107734

PPF extract inhibition of ROS production in UV-irradiated cells. Notes: ( A ) NHDF cells were exposed to UV irradiation at 40 J for 180 seconds and treated with PPF for 24 hours. Cell lysates were analyzed by immunoblotting using anti-TNFR6, anti-c-Jun, anti-c-Fos, anti-pp38, or anti-p-JNK antibodies. Results are representative of three independent experiments. ( B ) Total and tyrosine-phosphorylated p38, JUK, actin, and TNFR6 in the immunoprecipitates were quantified by Western analyses. Bar heights are mean ± SD of three independent experiments. * P
Figure Legend Snippet: PPF extract inhibition of ROS production in UV-irradiated cells. Notes: ( A ) NHDF cells were exposed to UV irradiation at 40 J for 180 seconds and treated with PPF for 24 hours. Cell lysates were analyzed by immunoblotting using anti-TNFR6, anti-c-Jun, anti-c-Fos, anti-pp38, or anti-p-JNK antibodies. Results are representative of three independent experiments. ( B ) Total and tyrosine-phosphorylated p38, JUK, actin, and TNFR6 in the immunoprecipitates were quantified by Western analyses. Bar heights are mean ± SD of three independent experiments. * P

Techniques Used: Inhibition, Irradiation, Western Blot

Related Articles

Transduction:

Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase
Article Snippet: Monoclonal antibodies against c-Jun were obtained from Transduction Laboratories (Lexington, KY). .. Polyclonal antibodies against c-Jun and Sp1, protein A-agarose and agarose conjugated to Sp1 or c-Jun antibodies were from Santa Cruz Biotechnology.

Centrifugation:

Article Title: The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation
Article Snippet: Lysates were clarified by centrifugation and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. .. Blots were incubated with antibodies against p-p38 (1:1000, #4511S; Cell Signalling Technology, Danvers, MA), p38 (#sc-7972), phosphorylated extracellular signal–regulated kinase-1/2 (#sc-7383), phosphorylated c-Jun N-terminal kinase (#sc-6254) (1:500; all from Santa Cruz Biotechnology), and β-actin antibody (1:5000, #A544; Sigma), followed by a secondary antibody conjugated to horseradish peroxidase (1:20,000; Dako, Santa Clara, CA).

Article Title: Growth Factor Stimulation Induces Cell Survival by c-Jun?ATF2-dependent Activation of Bcl-XL *
Article Snippet: Following stimulation, the cells were lysed in radioimmune precipitation assay plus buffer (50 m m Tris-HCl, pH 7.2, 100 m m NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 50 m m NaF, 2 m m sodium orthovanadate, 1 m m dithiothreitol, anti-protease, and anti-phosphatase cocktails (Sigma), and cell debris was eliminated by centrifugation at 21,000 × g at 4 °C for 10 min. .. The following antibodies were used for immunoblotting: anti-Bcl-XL (antibody 2762; Cell Signaling), anti-c-Jun (antibody sc-45X), anti-SP1 (antibody sc-59), anti-ATF2 (antibody sc-187) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ATF2 Thr(P)69/71 (antibody 05-891; Upstate), and anti-β-actin (antibody A5441; Sigma).

Luciferase:

Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase
Article Snippet: The luciferase assay system was from Promega. .. Polyclonal antibodies against c-Jun and Sp1, protein A-agarose and agarose conjugated to Sp1 or c-Jun antibodies were from Santa Cruz Biotechnology.

Neutralization:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: In brief, HaCaT cells were treated with 10 μM curcumin or ethanol (vehicle) for 24 hrs, and then crosslinked by 1% formaldehyde for 10 min with following neutralization by adding glycine to a final concentration of 0.1 M for 5 min at room temperature. .. Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Electrophoresis:

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner
Article Snippet: For the supershift assay, 10 μg of rabbit anti-c-jun antibody (Santa Cruz Biotechnology) or nonspecific IgG was added to the reaction mixture, subsequent to the addition of the 32 P-labeled oligonucleotide probe, and the mixture was incubated for 45 min at room temperature. .. Complexes were resolved by electrophoresis as described for the gel shift assay.

Incubation:

Article Title: The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation
Article Snippet: .. Blots were incubated with antibodies against p-p38 (1:1000, #4511S; Cell Signalling Technology, Danvers, MA), p38 (#sc-7972), phosphorylated extracellular signal–regulated kinase-1/2 (#sc-7383), phosphorylated c-Jun N-terminal kinase (#sc-6254) (1:500; all from Santa Cruz Biotechnology), and β-actin antibody (1:5000, #A544; Sigma), followed by a secondary antibody conjugated to horseradish peroxidase (1:20,000; Dako, Santa Clara, CA). .. Histopathologic Scoring and Immunohistochemistry Cryosections of colon and ileum samples were stained with H & E and scored in blinded fashion on the basis of changes of the epithelium and cell infiltration, as previously described.

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: After 10 min incubation on ice, nuclei were harvested in MNase Digestion Buffer and re-suspended in IP Dilution Buffer containing protease/phosphatase inhibitors, and then sonicated to achieve fragmentation of DNA to 200–1000 base pairs in length. .. Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner
Article Snippet: .. For the supershift assay, 10 μg of rabbit anti-c-jun antibody (Santa Cruz Biotechnology) or nonspecific IgG was added to the reaction mixture, subsequent to the addition of the 32 P-labeled oligonucleotide probe, and the mixture was incubated for 45 min at room temperature. .. Complexes were resolved by electrophoresis as described for the gel shift assay.

Article Title: Activator Protein-1 Activation in Acute Lung Injury
Article Snippet: .. Membranes were incubated with the following antibodies in a 1:1000 dilution: polyclonal rabbit anti-rat c-fos (Ab-2) (Oncogene Research Products, Uniondale, NY), polyclonal rabbit anti-rat c-jun (H-79), polyclonal rabbit anti-rat jun-B (210), and polyclonal rabbit anti-rat jun-D (329) (Santa Cruz Biotechnology). .. After five washes in TBST, membranes were incubated in a 1:10,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham).

Article Title: Growth Factor Stimulation Induces Cell Survival by c-Jun?ATF2-dependent Activation of Bcl-XL *
Article Snippet: The following antibodies were used for immunoblotting: anti-Bcl-XL (antibody 2762; Cell Signaling), anti-c-Jun (antibody sc-45X), anti-SP1 (antibody sc-59), anti-ATF2 (antibody sc-187) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ATF2 Thr(P)69/71 (antibody 05-891; Upstate), and anti-β-actin (antibody A5441; Sigma). .. For immunoprecipitations, the protein extracts prepared as described above were incubated for 1 h at 4 °C with anti-ATF2 (sc-187) coupled to Dynabeads® protein A (Dynal Biotech, Oslo, Norway).

Amplification:

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1
Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology). .. For PCR of the rat GCLC promoter region across the AP-1 site, an equal amount of template DNA from the antibody-treated, no-antibody, and input samples was amplified using forward primer 5′-CCAGTATTCTCTTGGGAACCAAG-3′ (bp −437 to −413 relative to the ATG start codon) and reverse primer 5′-CACGGGCTTCCTACTTGCGAC-3′ (bp −234 to −213 relative to the ATG start codon).

Expressing:

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling
Article Snippet: To examine protein expression, western blot analysis was performed by separating proteins on a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were then transferred onto an Immobilon-P transfer membrane (Millipore Co., Billerica, MA, USA). .. The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Western Blot:

Article Title: EBV-LMP1 targeted DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via the JNKs/HIF-1 pathway in nasopharyngeal carcinoma
Article Snippet: Paragraph title: Western blotting analysis ... The study employed antibodies against LMP1 (M0897, DAKO, Carpinteria, CA), c-Jun (sc-1694, Santa Cruz, CA), phosphorylated c-Jun (Ser 73, sc-7981, Santa Cruz), JNKs (9252, Cell Signaling, Beverly, MA), phosphorylated JNKs (Thr183/Tyr185, 9251, Cell Signaling), HIF-1(sc-53546, Santa Cruz), VEGF (sc-152, Santa Cruz) and β-actin (sc-8432, Santa Cruz).

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure
Article Snippet: Paragraph title: Western blotting and protein level quantification ... The PVDF membranes were then blocked with 5% skim milk in 1×TBST for 1 h, followed by probing with the primary antibodies including anti-Smad2 (Abcam, Shanghai, China, #ab63576), anti-pSmad2 (Sigma-Aldrich, #AB3849-I), anti-Smad3 (Abcam, #ab40854), anti-pSmad3 (Abcam, #ab52903), anti-CtBP2 (Abcam, #ab128871), anti-c-Jun (Santa Cruz Biotechnology, #sc-166540), anti-c-FOS (Santa Cruz Biotechnology, #sc-81209), anti-p300 (Sigma-Aldrich, #P2859), anti-TGF-β (Sigma-Aldrich, #SAB4502954), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Sigma-Aldrich, #05419), and anti-GAPDH (Abcam, #ab181602).

Article Title: The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation
Article Snippet: Paragraph title: Western Blot ... Blots were incubated with antibodies against p-p38 (1:1000, #4511S; Cell Signalling Technology, Danvers, MA), p38 (#sc-7972), phosphorylated extracellular signal–regulated kinase-1/2 (#sc-7383), phosphorylated c-Jun N-terminal kinase (#sc-6254) (1:500; all from Santa Cruz Biotechnology), and β-actin antibody (1:5000, #A544; Sigma), followed by a secondary antibody conjugated to horseradish peroxidase (1:20,000; Dako, Santa Clara, CA).

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling
Article Snippet: Paragraph title: Western blot analysis ... The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Article Title: Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer
Article Snippet: Paragraph title: Western blot ... The primary antibodies were rabbit anti- E-cadherin, rabbit anti-N-cadherin, rabbit anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-MMP9, mouse anti-MMP2, mouse anti-JNK1, mouse anti-p-JNK, rabbit anti-c-Jun, mouse anti-p-c-Jun, mouse anti-FAK, mouse anti-β-actin (Santa Cruz Biotechnology), rabbit anti-Paxillin, and rabbit anti-p-Paxillin (Abcam, Cambridge, MA, USA).

Article Title: Activator Protein-1 Activation in Acute Lung Injury
Article Snippet: Paragraph title: Western Blot Analysis ... Membranes were incubated with the following antibodies in a 1:1000 dilution: polyclonal rabbit anti-rat c-fos (Ab-2) (Oncogene Research Products, Uniondale, NY), polyclonal rabbit anti-rat c-jun (H-79), polyclonal rabbit anti-rat jun-B (210), and polyclonal rabbit anti-rat jun-D (329) (Santa Cruz Biotechnology).

Allele-specific Oligonucleotide:

Article Title: The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis
Article Snippet: BAX siRNA (sc-29212), BAK siRNA (sc-29786), C/EBPβ siRNA (sc-44251), c-JUN siRNA (sc-29223), ERα siRNA (sc-29305), p53 siRNA (sc-29435), STAT1 siRNA (sc-44123), STAT4 siRNA (sc-36568), and XBP-1 siRNA (sc-38627) were bought from Santa Cruz Biotechnology (Dallas, TX). .. Transfection of siRNA, miRNA mimics, or miRNA ASO was conducted with Lipofectamine 2000.

Transfection:

Article Title: The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis
Article Snippet: BAX siRNA (sc-29212), BAK siRNA (sc-29786), C/EBPβ siRNA (sc-44251), c-JUN siRNA (sc-29223), ERα siRNA (sc-29305), p53 siRNA (sc-29435), STAT1 siRNA (sc-44123), STAT4 siRNA (sc-36568), and XBP-1 siRNA (sc-38627) were bought from Santa Cruz Biotechnology (Dallas, TX). .. Transfection of siRNA, miRNA mimics, or miRNA ASO was conducted with Lipofectamine 2000.

TCA Precipitation:

Article Title: Activator Protein-1 Activation in Acute Lung Injury
Article Snippet: Protein concentrations in nuclear extracts were determined by bicinchoninic acid assay with trichloroacetic acid precipitation using BSA as a reference standard (Pierce Co.). .. Membranes were incubated with the following antibodies in a 1:1000 dilution: polyclonal rabbit anti-rat c-fos (Ab-2) (Oncogene Research Products, Uniondale, NY), polyclonal rabbit anti-rat c-jun (H-79), polyclonal rabbit anti-rat jun-B (210), and polyclonal rabbit anti-rat jun-D (329) (Santa Cruz Biotechnology).

Concentration Assay:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: In brief, HaCaT cells were treated with 10 μM curcumin or ethanol (vehicle) for 24 hrs, and then crosslinked by 1% formaldehyde for 10 min with following neutralization by adding glycine to a final concentration of 0.1 M for 5 min at room temperature. .. Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Protease Inhibitor:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: After washing twice with cold PBS, cells were harvested in ice-cold PBS (containing protease inhibitor cocktail), and then the cell pellets were suspended in cold membrane extraction lysis buffer containing protease/phosphatase inhibitors. .. Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Cell Culture:

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure
Article Snippet: Western blotting and protein level quantification Renal biopsies and cultured cells were applied to isolate total protein extracts using 1×RIPA buffer (Sigma-Aldrich, #R0278). .. The PVDF membranes were then blocked with 5% skim milk in 1×TBST for 1 h, followed by probing with the primary antibodies including anti-Smad2 (Abcam, Shanghai, China, #ab63576), anti-pSmad2 (Sigma-Aldrich, #AB3849-I), anti-Smad3 (Abcam, #ab40854), anti-pSmad3 (Abcam, #ab52903), anti-CtBP2 (Abcam, #ab128871), anti-c-Jun (Santa Cruz Biotechnology, #sc-166540), anti-c-FOS (Santa Cruz Biotechnology, #sc-81209), anti-p300 (Sigma-Aldrich, #P2859), anti-TGF-β (Sigma-Aldrich, #SAB4502954), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Sigma-Aldrich, #05419), and anti-GAPDH (Abcam, #ab181602).

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling
Article Snippet: Western blot analysis AGS cells were cultured in 100-mm diameter dishes. .. The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Article Title: Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer
Article Snippet: Western blot Cultured cells were collected and solubilized using protein lysis buffer. .. The primary antibodies were rabbit anti- E-cadherin, rabbit anti-N-cadherin, rabbit anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-MMP9, mouse anti-MMP2, mouse anti-JNK1, mouse anti-p-JNK, rabbit anti-c-Jun, mouse anti-p-c-Jun, mouse anti-FAK, mouse anti-β-actin (Santa Cruz Biotechnology), rabbit anti-Paxillin, and rabbit anti-p-Paxillin (Abcam, Cambridge, MA, USA).

Imaging:

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure
Article Snippet: The PVDF membranes were then blocked with 5% skim milk in 1×TBST for 1 h, followed by probing with the primary antibodies including anti-Smad2 (Abcam, Shanghai, China, #ab63576), anti-pSmad2 (Sigma-Aldrich, #AB3849-I), anti-Smad3 (Abcam, #ab40854), anti-pSmad3 (Abcam, #ab52903), anti-CtBP2 (Abcam, #ab128871), anti-c-Jun (Santa Cruz Biotechnology, #sc-166540), anti-c-FOS (Santa Cruz Biotechnology, #sc-81209), anti-p300 (Sigma-Aldrich, #P2859), anti-TGF-β (Sigma-Aldrich, #SAB4502954), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Sigma-Aldrich, #05419), and anti-GAPDH (Abcam, #ab181602). .. After washing 5 times with 1×TBST, the membrane was probed with secondary antibodies, and the protein signals were detected using an ECL mixture (Sigma-Aldrich, #GERPN2209) and recorded by a ChemiDoc MP imaging system (Bio-Rad, #17001402).

Protein Concentration:

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure
Article Snippet: The protein concentration was determined using a NanoDrop, and 50 μg of protein of each sample was loaded into 10% SDS-PAGE gels, followed by transfer to PVDF membranes (Bio-Rad, Shanghai, China, #1620177). .. The PVDF membranes were then blocked with 5% skim milk in 1×TBST for 1 h, followed by probing with the primary antibodies including anti-Smad2 (Abcam, Shanghai, China, #ab63576), anti-pSmad2 (Sigma-Aldrich, #AB3849-I), anti-Smad3 (Abcam, #ab40854), anti-pSmad3 (Abcam, #ab52903), anti-CtBP2 (Abcam, #ab128871), anti-c-Jun (Santa Cruz Biotechnology, #sc-166540), anti-c-FOS (Santa Cruz Biotechnology, #sc-81209), anti-p300 (Sigma-Aldrich, #P2859), anti-TGF-β (Sigma-Aldrich, #SAB4502954), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Sigma-Aldrich, #05419), and anti-GAPDH (Abcam, #ab181602).

Polymerase Chain Reaction:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology). .. The DNA sequence of S100A7 PCR primers (330 bp) was as following: forward strain 5’-CTTCTGTGAGGGGCTGACCA-3’ and reverse strain 5’- TCTATGACCCCCACCGCTGA-3’.

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1
Article Snippet: Twenty microliters of the reversed soluble chromatin sample was removed and used as input control (total chromatin fraction) for final PCR. .. Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

Sonication:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: After 10 min incubation on ice, nuclei were harvested in MNase Digestion Buffer and re-suspended in IP Dilution Buffer containing protease/phosphatase inhibitors, and then sonicated to achieve fragmentation of DNA to 200–1000 base pairs in length. .. Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1
Article Snippet: The sonicated cell lysates were spun in a microcentrifuge at 13,000 × g for 10 min at 4°C. .. Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

Recombinant:

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts
Article Snippet: Reagents Recombinant human cytokines and anti-human IL-36γ were purchased from R & D Systems (Minneapolis, MN). .. Antibodies against phosphorylated c-Jun, NF-κB p65, phosphorylated IκBα were purchased from Santa Cruz.

Nucleic Acid Electrophoresis:

Article Title: The Food Additive Maltodextrin Promotes Endoplasmic Reticulum Stress–Driven Mucus Depletion and Exacerbates Intestinal Inflammation
Article Snippet: Lysates were clarified by centrifugation and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. .. Blots were incubated with antibodies against p-p38 (1:1000, #4511S; Cell Signalling Technology, Danvers, MA), p38 (#sc-7972), phosphorylated extracellular signal–regulated kinase-1/2 (#sc-7383), phosphorylated c-Jun N-terminal kinase (#sc-6254) (1:500; all from Santa Cruz Biotechnology), and β-actin antibody (1:5000, #A544; Sigma), followed by a secondary antibody conjugated to horseradish peroxidase (1:20,000; Dako, Santa Clara, CA).

Electrophoretic Mobility Shift Assay:

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner
Article Snippet: Paragraph title: Nuclear extracts and gel shift assays. ... For the supershift assay, 10 μg of rabbit anti-c-jun antibody (Santa Cruz Biotechnology) or nonspecific IgG was added to the reaction mixture, subsequent to the addition of the 32 P-labeled oligonucleotide probe, and the mixture was incubated for 45 min at room temperature.

Sequencing:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology). .. The DNA sequence of S100A7 PCR primers (330 bp) was as following: forward strain 5’-CTTCTGTGAGGGGCTGACCA-3’ and reverse strain 5’- TCTATGACCCCCACCGCTGA-3’.

Polyacrylamide Gel Electrophoresis:

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling
Article Snippet: To examine protein expression, western blot analysis was performed by separating proteins on a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were then transferred onto an Immobilon-P transfer membrane (Millipore Co., Billerica, MA, USA). .. The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Chromatin Immunoprecipitation:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: Paragraph title: ChIP (Chromatin immunoprecipitation) analysis ... Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity
Article Snippet: Paragraph title: Chromatin immunoprecipitation ... Chromatin prepared from each treatment group was immunoprecipitated with anti-ERβ ( ) anti-c-Fos, or anti-c-Jun (both from Santa Cruz Biotechnology), following the protocol described by Ghoshal et al . ( ).

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1
Article Snippet: Paragraph title: ChIP assay. ... Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

SDS Page:

Article Title: EBV-LMP1 targeted DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via the JNKs/HIF-1 pathway in nasopharyngeal carcinoma
Article Snippet: Protein samples (50 μg) were separated by 12% SDS-PAGE, transferred onto nylon membranes and immunoblotted with primary antibodies. .. The study employed antibodies against LMP1 (M0897, DAKO, Carpinteria, CA), c-Jun (sc-1694, Santa Cruz, CA), phosphorylated c-Jun (Ser 73, sc-7981, Santa Cruz), JNKs (9252, Cell Signaling, Beverly, MA), phosphorylated JNKs (Thr183/Tyr185, 9251, Cell Signaling), HIF-1(sc-53546, Santa Cruz), VEGF (sc-152, Santa Cruz) and β-actin (sc-8432, Santa Cruz).

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure
Article Snippet: The protein concentration was determined using a NanoDrop, and 50 μg of protein of each sample was loaded into 10% SDS-PAGE gels, followed by transfer to PVDF membranes (Bio-Rad, Shanghai, China, #1620177). .. The PVDF membranes were then blocked with 5% skim milk in 1×TBST for 1 h, followed by probing with the primary antibodies including anti-Smad2 (Abcam, Shanghai, China, #ab63576), anti-pSmad2 (Sigma-Aldrich, #AB3849-I), anti-Smad3 (Abcam, #ab40854), anti-pSmad3 (Abcam, #ab52903), anti-CtBP2 (Abcam, #ab128871), anti-c-Jun (Santa Cruz Biotechnology, #sc-166540), anti-c-FOS (Santa Cruz Biotechnology, #sc-81209), anti-p300 (Sigma-Aldrich, #P2859), anti-TGF-β (Sigma-Aldrich, #SAB4502954), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Sigma-Aldrich, #05419), and anti-GAPDH (Abcam, #ab181602).

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling
Article Snippet: To examine protein expression, western blot analysis was performed by separating proteins on a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were then transferred onto an Immobilon-P transfer membrane (Millipore Co., Billerica, MA, USA). .. The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Article Title: Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer
Article Snippet: The proteins were then separated by size using SDS-PAGE and transferred to polyvinyl difluoride membranes (Millipore). .. The primary antibodies were rabbit anti- E-cadherin, rabbit anti-N-cadherin, rabbit anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-MMP9, mouse anti-MMP2, mouse anti-JNK1, mouse anti-p-JNK, rabbit anti-c-Jun, mouse anti-p-c-Jun, mouse anti-FAK, mouse anti-β-actin (Santa Cruz Biotechnology), rabbit anti-Paxillin, and rabbit anti-p-Paxillin (Abcam, Cambridge, MA, USA).

Plasmid Preparation:

Article Title: Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves' Disease: Apparent Role of ADAM17
Article Snippet: Polyclonal anti-IGF-1Rα and an anti-c-Jun Abs were from Santa Cruz Biotechnology (Santa Cruz, CA) while mAbs against IGF-1Rα and anti-Grb2 Abs were from US Biological, Swampscott, MA. .. Polyclonal anti-IGF-1Rα and an anti-c-Jun Abs were from Santa Cruz Biotechnology (Santa Cruz, CA) while mAbs against IGF-1Rα and anti-Grb2 Abs were from US Biological, Swampscott, MA.

Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase
Article Snippet: Qiagen-tip 100 was from Qiagen (Hilden, Germany). β-Galactosidase plasmid driven by cytomegalovirus (pCMVβ) was from CLONTECH. .. Polyclonal antibodies against c-Jun and Sp1, protein A-agarose and agarose conjugated to Sp1 or c-Jun antibodies were from Santa Cruz Biotechnology.

Article Title: The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis
Article Snippet: To infect podocytes with plenti-CMV-LOC105374325, plenti-CMV-Bax, plenti-CMV-Bak, or plenti-CMV-C/EBPβ plasmid, the lentiviral stock was mixed with Polybrene (1 μg/ml) and added to podocytes. .. BAX siRNA (sc-29212), BAK siRNA (sc-29786), C/EBPβ siRNA (sc-44251), c-JUN siRNA (sc-29223), ERα siRNA (sc-29305), p53 siRNA (sc-29435), STAT1 siRNA (sc-44123), STAT4 siRNA (sc-36568), and XBP-1 siRNA (sc-38627) were bought from Santa Cruz Biotechnology (Dallas, TX).

Software:

Article Title: EBV-LMP1 targeted DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via the JNKs/HIF-1 pathway in nasopharyngeal carcinoma
Article Snippet: The study employed antibodies against LMP1 (M0897, DAKO, Carpinteria, CA), c-Jun (sc-1694, Santa Cruz, CA), phosphorylated c-Jun (Ser 73, sc-7981, Santa Cruz), JNKs (9252, Cell Signaling, Beverly, MA), phosphorylated JNKs (Thr183/Tyr185, 9251, Cell Signaling), HIF-1(sc-53546, Santa Cruz), VEGF (sc-152, Santa Cruz) and β-actin (sc-8432, Santa Cruz). .. The relative protein levels were quantified using ImageJ software (NIH).

Negative Control:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology). .. Normal rabbit IgG, included in PierceTM Magnetic ChIP Kit, was used in the negative control IP experiments.

Binding Assay:

Article Title: EBV-LMP1 targeted DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via the JNKs/HIF-1 pathway in nasopharyngeal carcinoma
Article Snippet: Binding of primary antibodies was detected using peroxidase conjugated secondary antibodies (Santa Cruz, CA) and developed with an enhanced chemiluminescence detection kit (Pierce ECL, Thermo Scientific, Pittsburgh, PA.). .. The study employed antibodies against LMP1 (M0897, DAKO, Carpinteria, CA), c-Jun (sc-1694, Santa Cruz, CA), phosphorylated c-Jun (Ser 73, sc-7981, Santa Cruz), JNKs (9252, Cell Signaling, Beverly, MA), phosphorylated JNKs (Thr183/Tyr185, 9251, Cell Signaling), HIF-1(sc-53546, Santa Cruz), VEGF (sc-152, Santa Cruz) and β-actin (sc-8432, Santa Cruz).

Article Title: Activator Protein-1 Activation in Acute Lung Injury
Article Snippet: Nonspecific binding sites were blocked with TBST (40 mmol/L Tris, pH 7.6, 300 mmol/L NaCl, 0.1% Tween 20) containing 5% nonfat dry milk for 12 hours at 4°C. .. Membranes were incubated with the following antibodies in a 1:1000 dilution: polyclonal rabbit anti-rat c-fos (Ab-2) (Oncogene Research Products, Uniondale, NY), polyclonal rabbit anti-rat c-jun (H-79), polyclonal rabbit anti-rat jun-B (210), and polyclonal rabbit anti-rat jun-D (329) (Santa Cruz Biotechnology).

Protein Binding:

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner
Article Snippet: Gel shift assays were performed by incubating 2 μg of the nuclear extracts for 20 min at room temperature with a 32 P-end-labeled DNA fragment containing the putative protein binding site (for AP-1, 5′CGC TTG ATG AGT CAG CCG GAA3′; Promega; [γ-32 P]ATP was from Amersham Life Science). .. For the supershift assay, 10 μg of rabbit anti-c-jun antibody (Santa Cruz Biotechnology) or nonspecific IgG was added to the reaction mixture, subsequent to the addition of the 32 P-labeled oligonucleotide probe, and the mixture was incubated for 45 min at room temperature.

Acid Assay:

Article Title: Activator Protein-1 Activation in Acute Lung Injury
Article Snippet: Protein concentrations in nuclear extracts were determined by bicinchoninic acid assay with trichloroacetic acid precipitation using BSA as a reference standard (Pierce Co.). .. Membranes were incubated with the following antibodies in a 1:1000 dilution: polyclonal rabbit anti-rat c-fos (Ab-2) (Oncogene Research Products, Uniondale, NY), polyclonal rabbit anti-rat c-jun (H-79), polyclonal rabbit anti-rat jun-B (210), and polyclonal rabbit anti-rat jun-D (329) (Santa Cruz Biotechnology).

Immunoprecipitation:

Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity
Article Snippet: .. Chromatin prepared from each treatment group was immunoprecipitated with anti-ERβ ( ) anti-c-Fos, or anti-c-Jun (both from Santa Cruz Biotechnology), following the protocol described by Ghoshal et al . ( ). .. An approximately 950-bp fragment of the PTPRO promoter (−822 to +132) was amplified from lymphocyte DNA and cloned at the Sma I/ Bgl II sites of pGL3-Basic vector (Promega Corp., Madison, WI) to generate the PTP-P-Luc vector.

Article Title: Growth Factor Stimulation Induces Cell Survival by c-Jun?ATF2-dependent Activation of Bcl-XL *
Article Snippet: Paragraph title: Protein Extracts, Immunoblotting, and Immunoprecipitation ... The following antibodies were used for immunoblotting: anti-Bcl-XL (antibody 2762; Cell Signaling), anti-c-Jun (antibody sc-45X), anti-SP1 (antibody sc-59), anti-ATF2 (antibody sc-187) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ATF2 Thr(P)69/71 (antibody 05-891; Upstate), and anti-β-actin (antibody A5441; Sigma).

Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1
Article Snippet: .. Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology). .. For PCR of the rat GCLC promoter region across the AP-1 site, an equal amount of template DNA from the antibody-treated, no-antibody, and input samples was amplified using forward primer 5′-CCAGTATTCTCTTGGGAACCAAG-3′ (bp −437 to −413 relative to the ATG start codon) and reverse primer 5′-CACGGGCTTCCTACTTGCGAC-3′ (bp −234 to −213 relative to the ATG start codon).

Lysis:

Article Title: Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7
Article Snippet: After washing twice with cold PBS, cells were harvested in ice-cold PBS (containing protease inhibitor cocktail), and then the cell pellets were suspended in cold membrane extraction lysis buffer containing protease/phosphatase inhibitors. .. Immunoprecipitations were carried out using anti-c-Jun (sc-1694; Santa Cruz Biotechnology) and anti-c-Fos (sc-253; Santa Cruz Biotechnology).

Article Title: Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling
Article Snippet: Cells were grown to 80% confluency and the medium was then replaced with SFM for 4 h. The medium was then replaced with fresh SFM containing Cf-GP (5, 10 or 20 μ g/ml) for 24 h. For collection, cells were washed with phosphate-buffered saline (PBS) and then added to extraction lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μ g/ml aprotinin, 1 μ g/ml leupeptin, 1 μ g/ml pepstatin A, 0.25% Na-deoxycholate and 1 mM PMSF). .. The transferred membrane was blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20), and then shaking with the indicated primary antibodies (diluted 1:1,000): anti-Wnt-1, anti-Frizzled, anti-LRP, anti-APC, anti-Axin, anti-GSK-3β, anti-β-catenin, anti-E-cadherin, anti-Snail, anti-LEF-1, anti-Tcf, anti-ICAM-1, anti-c-jun, anti-c-myc or anti-cyclin D from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Article Title: Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer
Article Snippet: Western blot Cultured cells were collected and solubilized using protein lysis buffer. .. The primary antibodies were rabbit anti- E-cadherin, rabbit anti-N-cadherin, rabbit anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-MMP9, mouse anti-MMP2, mouse anti-JNK1, mouse anti-p-JNK, rabbit anti-c-Jun, mouse anti-p-c-Jun, mouse anti-FAK, mouse anti-β-actin (Santa Cruz Biotechnology), rabbit anti-Paxillin, and rabbit anti-p-Paxillin (Abcam, Cambridge, MA, USA).

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  • 99
    Santa Cruz Biotechnology anti c jun
    AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in <t>anti-c-Jun</t> antibody-immunoprecipited candidates, but not in <t>anti-IgG</t> antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P
    Anti C Jun, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti c jun/product/Santa Cruz Biotechnology
    Average 99 stars, based on 85 article reviews
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    79
    Santa Cruz Biotechnology mouse anti phosphorylated jun kinase
    Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear <t>phosphorylated</t> <t>Jun-Kinase</t> (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) <t>Anti-BRDU</t> (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.
    Mouse Anti Phosphorylated Jun Kinase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phosphorylated jun kinase/product/Santa Cruz Biotechnology
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    91
    Santa Cruz Biotechnology c jun antibodies
    Effect of EGF on the interaction between <t>Sp1</t> oligonucleotides and c-Jun/Sp1. Nuclear extracts from EGF-treated cells were prepared and subjected to the assay for binding of the c-Jun/Sp1 complex to Sp1 consensus sites. The 32 P-radiolabeled Sp1 oligonucleotide (●) and Sp1 mutant SPM (□) were used as a probe for binding, respectively. For background control, protein A-agarose was used to substitute <t>anti-c-Jun</t> antibody–agarose conjugate in the assay (■). Values are means ± SEM of three determinations.
    C Jun Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c jun antibodies/product/Santa Cruz Biotechnology
    Average 91 stars, based on 2 article reviews
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    AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in anti-c-Jun antibody-immunoprecipited candidates, but not in anti-IgG antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P

    Journal: Oncotarget

    Article Title: The histidine-rich calcium binding protein (HRC) promotes tumor metastasis in hepatocellular carcinoma and is upregulated by SATB1

    doi:

    Figure Lengend Snippet: AP-1 is critical for SATB1-mediated HRC expression (A) Luciferase activity assay showed that SATB1 significantly increased AP-1 activity in SMMC-7721 and HEK 293T cells. (B) Overexpression of c-Jun enhanced while knockdown of c-Jun suppressed HRC promoter activity. Data are represented as the mean ± SD. (C) EMSA and (D) ChIP assay showed a direct binding of AP-1 to the HRC promoter. (C) The shift bands showed AP-1 combined with HRC promoter and this binding activity could be blocked by unlabeled AP-1 probe but mutant AP-1 probe. Supershift band showed c-Jun antibody blocked the mobility of the bands. (D) PCR showed HRC promoter could be detected in anti-c-Jun antibody-immunoprecipited candidates, but not in anti-IgG antibody-immunoprecipited candidates. (E) and (F) The effect of silencing endogenous c-Jun on SATB1-induced HRC promoter activation (E) and expression (F) Knockdown of c-Jun significantly abolished SATB1-induced HRC promoter activation and expression.* P

    Article Snippet: The purified chromatin was immunoprecipitated using 2 μg of anti-c-Jun, or irrelevant antibody anti-IgG (Santa Cruz).

    Techniques: Expressing, Luciferase, Activity Assay, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Mutagenesis, Polymerase Chain Reaction, Activation Assay

    Effect of TBH on electrophoretic mobility shift and supershift assays for AP-1 binding. Nuclear protein extracts (10 μg) were obtained from WT, F1, and F2 cells treated with TBH (60 μM for 0, 4, or 8 h), and EMSA was done as described in Materials and Methods using a consensus AP-1 probe. Panel A shows supershift analysis using anti-c-Jun antibodies, and panel B shows supershift analysis using anti-c-Fos antibodies. The arrows to the right point to complexes that were supershifted in the presence of specific antibodies. Representative EMSAs are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

    doi: 10.1128/MCB.25.14.5933-5946.2005

    Figure Lengend Snippet: Effect of TBH on electrophoretic mobility shift and supershift assays for AP-1 binding. Nuclear protein extracts (10 μg) were obtained from WT, F1, and F2 cells treated with TBH (60 μM for 0, 4, or 8 h), and EMSA was done as described in Materials and Methods using a consensus AP-1 probe. Panel A shows supershift analysis using anti-c-Jun antibodies, and panel B shows supershift analysis using anti-c-Fos antibodies. The arrows to the right point to complexes that were supershifted in the presence of specific antibodies. Representative EMSAs are shown.

    Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay

    EMSA and supershift analysis of the rat GCLC AP-1 and NF-κB sites. WT cells were treated with TBH (60 μM for 8 h) or vehicle control and subjected to EMSA with supershift analysis for the AP-1 site at −356 or the NF-κB site at −378. Supershift analysis was performed using antibodies directed against c-Jun, Nrf1, and Nrf2 for the AP-1 site (A) and p50, Nrf1, and Nrf2 for the NF-κB site (B). Note that supershift occurred only with anti-c-Jun antibodies for the AP-1 site and anti-p50 antibodies for the NF-κB site. As a positive control, TBH treatment induced Nrf1 and Nrf2 binding to the ARE site of the mouse GCLM (C). Arrows in panel C point to the Nrf1 and Nrf2 supershifts.

    Journal: Molecular and Cellular Biology

    Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

    doi: 10.1128/MCB.25.14.5933-5946.2005

    Figure Lengend Snippet: EMSA and supershift analysis of the rat GCLC AP-1 and NF-κB sites. WT cells were treated with TBH (60 μM for 8 h) or vehicle control and subjected to EMSA with supershift analysis for the AP-1 site at −356 or the NF-κB site at −378. Supershift analysis was performed using antibodies directed against c-Jun, Nrf1, and Nrf2 for the AP-1 site (A) and p50, Nrf1, and Nrf2 for the NF-κB site (B). Note that supershift occurred only with anti-c-Jun antibodies for the AP-1 site and anti-p50 antibodies for the NF-κB site. As a positive control, TBH treatment induced Nrf1 and Nrf2 binding to the ARE site of the mouse GCLM (C). Arrows in panel C point to the Nrf1 and Nrf2 supershifts.

    Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

    Techniques: Positive Control, Binding Assay

    Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-?B and AP-1

    doi: 10.1128/MCB.25.14.5933-5946.2005

    Figure Lengend Snippet: Steady-state protein levels of the AP-1 family members in WT, F1, and F2 cells. Total cell lysates (40 μg/lane) from WT, F1, and F2 cells were subjected to Western blot analysis using anti-c-Fos, c-Jun, phospho-c-Jun (p-c-Jun), JunB, JunD, Fra-1, Fra-2, and JAB1 antibodies as described in Materials and Methods. The same membranes were stripped and probed with antibodies against actin to ensure equal protein loading. The right panels show densitometric changes expressed as percentages of WT. *, P

    Article Snippet: Antibodies used for immunoprecipitation were anti-c-Jun, c-Fos, Fra-1, and Nrf2 antibodies (Santa Cruz Biotechnology).

    Techniques: Western Blot

    Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear phosphorylated Jun-Kinase (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) Anti-BRDU (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.

    Journal: PLoS ONE

    Article Title: Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster

    doi: 10.1371/journal.pone.0025087

    Figure Lengend Snippet: Germ line cells from nup98-96 2288 /Df(3R)mbc-R1 mutant animals have normal protein localization patterns. Immuno-labelling of germaria, antibodies and genotypes as indicated. (A, B) nuclear Groucho (red) and cytoplasmic Vasa (green); (C, D) nuclear phosphorylated Jun-Kinase (red) and cytoplasmic Vasa (green); note that some germaria (arrowheads) are empty in the nup98-96 2288 /Df(3R)mbc-R1 mutant ovaries; (E, F) cytoplasmic Sex-lethal in GSCs and gonialblasts; (G, H) nuclear phosphorylated Histone-H3 (red) and cytoplasmic Vasa (green); (I, J) Anti-BRDU (green) and DAPI (red). Asterisks: apical tips, arrows point to intra-cellular protein localizations, scale bars: 50 µm.

    Article Snippet: Goat anti-Vasa (1∶1000) and mouse anti-phosphorylated Jun-Kinase (1∶50) were obtained from Santa Cruz Biotechnology.

    Techniques: Mutagenesis

    Effect of EGF on the interaction between Sp1 oligonucleotides and c-Jun/Sp1. Nuclear extracts from EGF-treated cells were prepared and subjected to the assay for binding of the c-Jun/Sp1 complex to Sp1 consensus sites. The 32 P-radiolabeled Sp1 oligonucleotide (●) and Sp1 mutant SPM (□) were used as a probe for binding, respectively. For background control, protein A-agarose was used to substitute anti-c-Jun antibody–agarose conjugate in the assay (■). Values are means ± SEM of three determinations.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

    doi:

    Figure Lengend Snippet: Effect of EGF on the interaction between Sp1 oligonucleotides and c-Jun/Sp1. Nuclear extracts from EGF-treated cells were prepared and subjected to the assay for binding of the c-Jun/Sp1 complex to Sp1 consensus sites. The 32 P-radiolabeled Sp1 oligonucleotide (●) and Sp1 mutant SPM (□) were used as a probe for binding, respectively. For background control, protein A-agarose was used to substitute anti-c-Jun antibody–agarose conjugate in the assay (■). Values are means ± SEM of three determinations.

    Article Snippet: Polyclonal antibodies against c-Jun and Sp1, protein A-agarose and agarose conjugated to Sp1 or c-Jun antibodies were from Santa Cruz Biotechnology.

    Techniques: Binding Assay, Mutagenesis

    Binding of c-Jun and Sp1 in cells overexpressing c-Jun or Ha-ras. Cells were transfected with a different amount of pRSVjun or pSV2ras by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun protein ( A ) and the coimmunoprecipitated c-Jun/Sp1 complex by using anti-Sp1 antibodies ( B and C ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

    doi:

    Figure Lengend Snippet: Binding of c-Jun and Sp1 in cells overexpressing c-Jun or Ha-ras. Cells were transfected with a different amount of pRSVjun or pSV2ras by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun protein ( A ) and the coimmunoprecipitated c-Jun/Sp1 complex by using anti-Sp1 antibodies ( B and C ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies.

    Article Snippet: Polyclonal antibodies against c-Jun and Sp1, protein A-agarose and agarose conjugated to Sp1 or c-Jun antibodies were from Santa Cruz Biotechnology.

    Techniques: Binding Assay, Transfection, Incubation, Expressing, Western Blot

    Effect of c-Jun dominant negative mutant on c-Jun/Sp1 interaction in pRSVjun-treated cells. Cells were transfected with 2 μg of pRSVjun and a different amount of c-Jun dominant negative vector TAM-67 by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun and TAM-67 proteins ( A ) and the coimmunoprecipitated c-Jun/Sp1 and TAM-67/Sp1 complex by using anti-Sp1 antibodies ( B ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies. pcDNA3.1 was used as a vector to adjust for the same amount of plasmids in each experiment.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

    doi:

    Figure Lengend Snippet: Effect of c-Jun dominant negative mutant on c-Jun/Sp1 interaction in pRSVjun-treated cells. Cells were transfected with 2 μg of pRSVjun and a different amount of c-Jun dominant negative vector TAM-67 by the lipofection method. After the change of Opti-MEM medium to 3 ml of fresh culture medium in a 6-cm plastic dish, cells were incubated for an additional 36 h. Expression of c-Jun and TAM-67 proteins ( A ) and the coimmunoprecipitated c-Jun/Sp1 and TAM-67/Sp1 complex by using anti-Sp1 antibodies ( B ) was analyzed by Western blot with anti-c-Jun and anti-Sp1 antibodies. pcDNA3.1 was used as a vector to adjust for the same amount of plasmids in each experiment.

    Article Snippet: Polyclonal antibodies against c-Jun and Sp1, protein A-agarose and agarose conjugated to Sp1 or c-Jun antibodies were from Santa Cruz Biotechnology.

    Techniques: Dominant Negative Mutation, Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot