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rabbit polyclonal anti-nf-κb p65 protein subunit (c-20) (Santa Cruz Biotechnology)

Name: rabbit polyclonal anti-nf-κb p65 protein subunit (c-20)
Brand: Santa Cruz Biotechnology
Rating: 90 (11893 reviews)

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Santa Cruz Biotechnology rabbit polyclonal anti-nf-κb p65 protein subunit (c-20)

Microarray sections of non-malignant and malignant prostate tissue specimens were probed with an anti-EGFR, -Ser 473 <t>-pAkt,</t> <t>-NF-κB</t> <t>p65</t> or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained tissue specimens of normal prostate, non-malignant adjacent tissues of prostatic tumor and prostatic adenocarcinoma obtained for ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 are shown at original magnifications of ×100 and ×400. The arrows indicate the localization of basal cells in normal and non-malignant prostate epithelium and immunostaining detected for these biomarkers in prostatic adenocarcinoma tissue specimen. Moreover, the positive immunostaining detected for secreted MIC-1 protein in the stromal compartment adjacent to prostatic tumor tissue is also indicated.

Rabbit Polyclonal Anti Nf κb P65 Protein Subunit (C 20), supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 11893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal anti-nf-κb p65 protein subunit (c-20)/product/Santa Cruz Biotechnology
Average 90 stars, based on 11893 article reviews

rabbit polyclonal anti-nf-κb p65 protein subunit (c-20) - by Bioz Stars, 2026-03

90/100 stars

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1) Product Images from "Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies"

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031919

Microarray sections of non-malignant and malignant prostate tissue specimens were probed with an anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained tissue specimens of normal prostate, non-malignant adjacent tissues of prostatic tumor and prostatic adenocarcinoma obtained for ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 are shown at original magnifications of ×100 and ×400. The arrows indicate the localization of basal cells in normal and non-malignant prostate epithelium and immunostaining detected for these biomarkers in prostatic adenocarcinoma tissue specimen. Moreover, the positive immunostaining detected for secreted MIC-1 protein in the stromal compartment adjacent to prostatic tumor tissue is also indicated.

Figure Legend Snippet: Microarray sections of non-malignant and malignant prostate tissue specimens were probed with an anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained tissue specimens of normal prostate, non-malignant adjacent tissues of prostatic tumor and prostatic adenocarcinoma obtained for ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 are shown at original magnifications of ×100 and ×400. The arrows indicate the localization of basal cells in normal and non-malignant prostate epithelium and immunostaining detected for these biomarkers in prostatic adenocarcinoma tissue specimen. Moreover, the positive immunostaining detected for secreted MIC-1 protein in the stromal compartment adjacent to prostatic tumor tissue is also indicated.

Techniques Used: Microarray, Blocking Assay, Microscopy, Staining, Immunostaining

Box plots showing the expression levels of ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 during PC progression to metastatic disease stages. *, P <0.0001, indicates a significant increase between the composite scores obtained for prostatic adenocarcinoma and PC bone metastasis specimens relative to composite scores obtained for normal prostatic tissues.

Figure Legend Snippet: Box plots showing the expression levels of ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 during PC progression to metastatic disease stages. *, P <0.0001, indicates a significant increase between the composite scores obtained for prostatic adenocarcinoma and PC bone metastasis specimens relative to composite scores obtained for normal prostatic tissues.

Techniques Used: Expressing

Microarray sections of PC bone metastasis tissue specimens were probed with anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained PC bone tissue specimens obtained for EGFR, Ser 473 -pAkt, NF-κB p65 or MIC-1 are shown at original magnifications of ×100 and ×400.

Figure Legend Snippet: Microarray sections of PC bone metastasis tissue specimens were probed with anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained PC bone tissue specimens obtained for EGFR, Ser 473 -pAkt, NF-κB p65 or MIC-1 are shown at original magnifications of ×100 and ×400.

Techniques Used: Microarray, Blocking Assay, Microscopy, Staining

The double immunofluorescence analyses of the co-localization of the expression of markers in normal prostate and prostatic adenocarcinoma specimens from patients was simultaneously done with fluorescein-labeled anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody (green) plus phycoerythrin-labeled anti-CD133 antibody (red) after blocking with goat serum as described in . The arrows indicate a double staining (yellow/purple) detected by confocal analyses, which is indicative of the co-localization of these markers. Representative pictures are shown at the original magnification of ×630.

Figure Legend Snippet: The double immunofluorescence analyses of the co-localization of the expression of markers in normal prostate and prostatic adenocarcinoma specimens from patients was simultaneously done with fluorescein-labeled anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody (green) plus phycoerythrin-labeled anti-CD133 antibody (red) after blocking with goat serum as described in . The arrows indicate a double staining (yellow/purple) detected by confocal analyses, which is indicative of the co-localization of these markers. Representative pictures are shown at the original magnification of ×630.

Techniques Used: Immunofluorescence, Expressing, Labeling, Blocking Assay, Double Staining

$a ) Representative data of the Hoechst dye efflux profile obtained after staining of parental WPE1-NB26 cell line with fluorescent Hoechst dye showing the SP cell subpopulation (green) and non-SP fraction (blue) detected in the total mass of parental WPE1-NB26 cells. b ) FACS profiles obtained after staining of parental WPE1-NB26 cells with phycoerythrin -labeled anti-CD133 antibody showing the percentage of CD133 + and CD133 − PC cells detected in the total mass of parental WPE1-NB26 cells. c ) Comparative Western blot analyses of expression levels of prostatic stem cell-like markers (CD133 and CD44), multidrug transporter ABCG2, EGFR, Ser 473 -pAkt, NF-κB p65 subunit and secreted MIC-1 proteins detected in the SP and non-SP cell fractions isolated from parental WPE1-NB26 cell line. d ) Immunofluorescence staining of methanol-fixed prostaspheres derived from SP cells and the adherent non-SP cell fraction isolated from the parental WPE1-NB26 cell line were done with anti-EGFR plus Tyr 1173 -pEGFR, Ser 473 -pAkt, NF-κB p65 or MIC-1 primary antibody plus fluorescein (green) and/or Texas red secondary antibody (red) and 4′,6-diamidino-2-phenylindole (nuclear blue) after blocking with goat serum. Representative pictures showing the expression level and cellular localization obtained for the stem cell-like markers including CD133 (red), CD44 (green) and ABCG2 (red) as well as overlaps of EGFR/Tyr 1173 -pEGFR (red/green, hybrid yellow), Ser 473 -pAkt (red), NF-κB p65 (red) and MIC-1 (red) are shown at the original magnification of ×630.$
Figure Legend Snippet: a ) Representative data of the Hoechst dye efflux profile obtained after staining of parental WPE1-NB26 cell line with fluorescent Hoechst dye showing the SP cell subpopulation (green) and non-SP fraction (blue) detected in the total mass of parental WPE1-NB26 cells. b ) FACS profiles obtained after staining of parental WPE1-NB26 cells with phycoerythrin -labeled anti-CD133 antibody showing the percentage of CD133 + and CD133 − PC cells detected in the total mass of parental WPE1-NB26 cells. c ) Comparative Western blot analyses of expression levels of prostatic stem cell-like markers (CD133 and CD44), multidrug transporter ABCG2, EGFR, Ser 473 -pAkt, NF-κB p65 subunit and secreted MIC-1 proteins detected in the SP and non-SP cell fractions isolated from parental WPE1-NB26 cell line. d ) Immunofluorescence staining of methanol-fixed prostaspheres derived from SP cells and the adherent non-SP cell fraction isolated from the parental WPE1-NB26 cell line were done with anti-EGFR plus Tyr 1173 -pEGFR, Ser 473 -pAkt, NF-κB p65 or MIC-1 primary antibody plus fluorescein (green) and/or Texas red secondary antibody (red) and 4′,6-diamidino-2-phenylindole (nuclear blue) after blocking with goat serum. Representative pictures showing the expression level and cellular localization obtained for the stem cell-like markers including CD133 (red), CD44 (green) and ABCG2 (red) as well as overlaps of EGFR/Tyr 1173 -pEGFR (red/green, hybrid yellow), Ser 473 -pAkt (red), NF-κB p65 (red) and MIC-1 (red) are shown at the original magnification of ×630.

Techniques Used: Staining, Labeling, Western Blot, Expressing, Isolation, Immunofluorescence, Derivative Assay, Blocking Assay

$The SP and non-SP cell fractions from the WPE1-NB26 cell line were subjected to the prostasphere formation culture on an ultra-low attachment plate in serum-free keratinocyte medium. The representative pictures of the dense prostaspheres formed by SP WPE1-NB26 cells ( a ) without or ( b ) after a treatment with exogenous EGF as compared to diffuse, abortive and very small aggregates formed by non-SP WPE1-NB26 cells are shown at a similar magnification of ×200. Moreover, the representative pictures of the prostaspheres formed by the SP WPE1-NB26 cell fraction ( a ) without or ( b ) after a treatment with exogenous EGF in the presence of different drugs, including a specific inhibitory agent of EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide), MIC-1 (anti-MIC-1 antibody) or docetaxel, are also shown at a similar magnification of ×200. The quantitative data of the number of prostaspheres formed by the SP WPE1-NB26 cell fraction ( c ) without or ( d ) after a treatment with exogenous EGF in the absence (control) or presence of different inhibitory agents obtained from at least 3 separate experiments are shown.$
Figure Legend Snippet: The SP and non-SP cell fractions from the WPE1-NB26 cell line were subjected to the prostasphere formation culture on an ultra-low attachment plate in serum-free keratinocyte medium. The representative pictures of the dense prostaspheres formed by SP WPE1-NB26 cells ( a ) without or ( b ) after a treatment with exogenous EGF as compared to diffuse, abortive and very small aggregates formed by non-SP WPE1-NB26 cells are shown at a similar magnification of ×200. Moreover, the representative pictures of the prostaspheres formed by the SP WPE1-NB26 cell fraction ( a ) without or ( b ) after a treatment with exogenous EGF in the presence of different drugs, including a specific inhibitory agent of EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide), MIC-1 (anti-MIC-1 antibody) or docetaxel, are also shown at a similar magnification of ×200. The quantitative data of the number of prostaspheres formed by the SP WPE1-NB26 cell fraction ( c ) without or ( d ) after a treatment with exogenous EGF in the absence (control) or presence of different inhibitory agents obtained from at least 3 separate experiments are shown.

Techniques Used:

$a ) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b ) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c ) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d ) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P <0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.$
Figure Legend Snippet: a ) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b ) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c ) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d ) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P <0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.

Techniques Used: Derivative Assay, Immunofluorescence, Staining, Expressing, TUNEL Assay

$The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P <0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.$
Figure Legend Snippet: The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P <0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.

Techniques Used:

Image Search Results

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: Microarray sections of non-malignant and malignant prostate tissue specimens were probed with an anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained tissue specimens of normal prostate, non-malignant adjacent tissues of prostatic tumor and prostatic adenocarcinoma obtained for ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 are shown at original magnifications of ×100 and ×400. The arrows indicate the localization of basal cells in normal and non-malignant prostate epithelium and immunostaining detected for these biomarkers in prostatic adenocarcinoma tissue specimen. Moreover, the positive immunostaining detected for secreted MIC-1 protein in the stromal compartment adjacent to prostatic tumor tissue is also indicated.

Article Snippet: The rabbit polyclonal anti-CD133 antibody (H-284), mouse monoclonal anti-CD44 (HCAM, F-4) antibody, rabbit polyclonal anti-ABCG2 antibody (B-25), rabbit polyclonal anti-EGFR antibody (1005), goat polyclonal anti-Tyr 1173 -phospho-EGFR antibody (1173) recognizing the EGFR form phosphorylated at tyrosine 1173 and rabbit polyclonal anti-NF-κB p65 protein subunit (C-20) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Microarray, Blocking Assay, Microscopy, Staining, Immunostaining

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: Box plots showing the expression levels of ( a ) EGFR, ( b ) Ser 473 -pAkt, ( c ) NF-κB p65 and ( d ) MIC-1 during PC progression to metastatic disease stages. *, P <0.0001, indicates a significant increase between the composite scores obtained for prostatic adenocarcinoma and PC bone metastasis specimens relative to composite scores obtained for normal prostatic tissues.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: Microarray sections of PC bone metastasis tissue specimens were probed with anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained PC bone tissue specimens obtained for EGFR, Ser 473 -pAkt, NF-κB p65 or MIC-1 are shown at original magnifications of ×100 and ×400.

Techniques: Microarray, Blocking Assay, Microscopy, Staining

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: The double immunofluorescence analyses of the co-localization of the expression of markers in normal prostate and prostatic adenocarcinoma specimens from patients was simultaneously done with fluorescein-labeled anti-EGFR, -Ser 473 -pAkt, -NF-κB p65 or -MIC-1 antibody (green) plus phycoerythrin-labeled anti-CD133 antibody (red) after blocking with goat serum as described in . The arrows indicate a double staining (yellow/purple) detected by confocal analyses, which is indicative of the co-localization of these markers. Representative pictures are shown at the original magnification of ×630.

Techniques: Immunofluorescence, Expressing, Labeling, Blocking Assay, Double Staining

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: a ) Representative data of the Hoechst dye efflux profile obtained after staining of parental WPE1-NB26 cell line with fluorescent Hoechst dye showing the SP cell subpopulation (green) and non-SP fraction (blue) detected in the total mass of parental WPE1-NB26 cells. b ) FACS profiles obtained after staining of parental WPE1-NB26 cells with phycoerythrin -labeled anti-CD133 antibody showing the percentage of CD133 + and CD133 − PC cells detected in the total mass of parental WPE1-NB26 cells. c ) Comparative Western blot analyses of expression levels of prostatic stem cell-like markers (CD133 and CD44), multidrug transporter ABCG2, EGFR, Ser 473 -pAkt, NF-κB p65 subunit and secreted MIC-1 proteins detected in the SP and non-SP cell fractions isolated from parental WPE1-NB26 cell line. d ) Immunofluorescence staining of methanol-fixed prostaspheres derived from SP cells and the adherent non-SP cell fraction isolated from the parental WPE1-NB26 cell line were done with anti-EGFR plus Tyr 1173 -pEGFR, Ser 473 -pAkt, NF-κB p65 or MIC-1 primary antibody plus fluorescein (green) and/or Texas red secondary antibody (red) and 4′,6-diamidino-2-phenylindole (nuclear blue) after blocking with goat serum. Representative pictures showing the expression level and cellular localization obtained for the stem cell-like markers including CD133 (red), CD44 (green) and ABCG2 (red) as well as overlaps of EGFR/Tyr 1173 -pEGFR (red/green, hybrid yellow), Ser 473 -pAkt (red), NF-κB p65 (red) and MIC-1 (red) are shown at the original magnification of ×630.

Techniques: Staining, Labeling, Western Blot, Expressing, Isolation, Immunofluorescence, Derivative Assay, Blocking Assay

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: The SP and non-SP cell fractions from the WPE1-NB26 cell line were subjected to the prostasphere formation culture on an ultra-low attachment plate in serum-free keratinocyte medium. The representative pictures of the dense prostaspheres formed by SP WPE1-NB26 cells ( a ) without or ( b ) after a treatment with exogenous EGF as compared to diffuse, abortive and very small aggregates formed by non-SP WPE1-NB26 cells are shown at a similar magnification of ×200. Moreover, the representative pictures of the prostaspheres formed by the SP WPE1-NB26 cell fraction ( a ) without or ( b ) after a treatment with exogenous EGF in the presence of different drugs, including a specific inhibitory agent of EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide), MIC-1 (anti-MIC-1 antibody) or docetaxel, are also shown at a similar magnification of ×200. The quantitative data of the number of prostaspheres formed by the SP WPE1-NB26 cell fraction ( c ) without or ( d ) after a treatment with exogenous EGF in the absence (control) or presence of different inhibitory agents obtained from at least 3 separate experiments are shown.

Techniques:

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: a ) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b ) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c ) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d ) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P <0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.

Techniques: Derivative Assay, Immunofluorescence, Staining, Expressing, TUNEL Assay

Journal: PLoS ONE

Article Title: Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

doi: 10.1371/journal.pone.0031919

Figure Lengend Snippet: The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P <0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.

Techniques:

Effect of porphyrins on protein RAS and ERK pathway . (A) Immunoblots showing the expression level of protein RAS in melanoma B78-H1 cells 24 h after treatment with porphyrins P2, P4 and C14 (1 and 5 μM) without irradiation. C14 at 5 μM reduces the level of protein RAS through a light-independent mechanism; (B) Immunoblots showing the levels of RAS, ERK, P-ERK, NF-κB and β-actin in untreated or porphyrin/light treated B78-H1 cells, 24 h after light treatment.

Journal: Molecular Cancer

Article Title: Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies

doi: 10.1186/1476-4598-13-75

Figure Lengend Snippet: Effect of porphyrins on protein RAS and ERK pathway . (A) Immunoblots showing the expression level of protein RAS in melanoma B78-H1 cells 24 h after treatment with porphyrins P2, P4 and C14 (1 and 5 μM) without irradiation. C14 at 5 μM reduces the level of protein RAS through a light-independent mechanism; (B) Immunoblots showing the levels of RAS, ERK, P-ERK, NF-κB and β-actin in untreated or porphyrin/light treated B78-H1 cells, 24 h after light treatment.

Article Snippet: Total protein lysates (30 μg), obtained 24 h after porphyrin/light treatment (see above), were run on 12% SDS-PAGE and blotted to a nitrocellulose membrane 70 V for 2 h. The membrane was treated for 1 h with PBS-0.01% Tween (Sigma-Aldrich, Milan, Italy) containing 5% dry non-fat-milk and incubated overnight at 4°C with the primary antibodies: mouse monoclonal c-KRAS Oncogene, (Cell Signaling, Merck Millipore, Darmstadt, Germany) diluted 1:40, rabbit polyclonal anti-NF-κB p65 (C-20, sc-372 Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:1000; rabbit polyclonal anti-ERK (p44/42 MAPK, 9102, Cell Signalling, Danvers MA) diluted 1:1000; rabbit polyclonal anti-P-ERK (phospho-p 44/42 MAPK, 9101Cell Signalling, Danvers MA) diluted 1:1000; rabbit polyclonal anti-PARP (9542, Cell Signalling, Danvers MA) diluted 1:1000. β-Actin was used as an internal control.

Techniques: Western Blot, Expressing, Irradiation

( A ) RT-qPCR results show the CALCB mRNA levels at 0 hr, 12 hr, and 12 days in TNF-α-treated WT and RELA KO cells. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. Two-tailed t -test. ( B ) RT-qPCR results show the transcriptional memory of CALCB in WT and RELA KO cells in response to TNF-α. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. One-tailed t -test. ( C ) Sandwich ELISA results show the CGRP release level in the media for WT and RELA KO cells in response to TNF-α. Data are shown as the mean from two independent experiments. One-tailed t -test. ( D ) RT-qPCR results show the CALCB mRNA levels in WT cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. ( E ) RT-qPCR results show the CALCB mRNA level in WT cells with various pretreatments that received a second TNF-α induction for 12 hr. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. ( F ) RT-qPCR results show the CALCB mRNA level in WT, TET1 KO, TET2 KO, TET3 KO, and TET TKO cells treated with TNF-α stimulation for 0 hr, 12 hr, and 12 days. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. Two-tailed t -test. ( G ) RT-qPCR results show the transcriptional memory of CALCB in WT, TET1 KO, TET2 KO, TET3 KO, and TET TKO cells in response to TNF-α. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. One-tailed t -test. ( H ) Expression levels (in FPKM) of EGFP , CALCB , PTGES , LHX2 , TNF , CCL2 for WT, and TET TKO cell in various treatment conditions. Data are shown as the mean from two independent experiments. One-tailed t -test. Figure 3—source data 1. Related to . Figure 3—source data 2. Related to . Figure 3—source data 3. Related to . Figure 3—source data 4. Related to . Figure 3—source data 5. Related to . Figure 3—source data 6. Related to . Figure 3—source data 7. Related to . Figure 3—source data 8. Related to .

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet: ( A ) RT-qPCR results show the CALCB mRNA levels at 0 hr, 12 hr, and 12 days in TNF-α-treated WT and RELA KO cells. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. Two-tailed t -test. ( B ) RT-qPCR results show the transcriptional memory of CALCB in WT and RELA KO cells in response to TNF-α. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. One-tailed t -test. ( C ) Sandwich ELISA results show the CGRP release level in the media for WT and RELA KO cells in response to TNF-α. Data are shown as the mean from two independent experiments. One-tailed t -test. ( D ) RT-qPCR results show the CALCB mRNA levels in WT cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. ( E ) RT-qPCR results show the CALCB mRNA level in WT cells with various pretreatments that received a second TNF-α induction for 12 hr. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. ( F ) RT-qPCR results show the CALCB mRNA level in WT, TET1 KO, TET2 KO, TET3 KO, and TET TKO cells treated with TNF-α stimulation for 0 hr, 12 hr, and 12 days. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. Two-tailed t -test. ( G ) RT-qPCR results show the transcriptional memory of CALCB in WT, TET1 KO, TET2 KO, TET3 KO, and TET TKO cells in response to TNF-α. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. One-tailed t -test. ( H ) Expression levels (in FPKM) of EGFP , CALCB , PTGES , LHX2 , TNF , CCL2 for WT, and TET TKO cell in various treatment conditions. Data are shown as the mean from two independent experiments. One-tailed t -test. Figure 3—source data 1. Related to . Figure 3—source data 2. Related to . Figure 3—source data 3. Related to . Figure 3—source data 4. Related to . Figure 3—source data 5. Related to . Figure 3—source data 6. Related to . Figure 3—source data 7. Related to . Figure 3—source data 8. Related to .

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: Quantitative RT-PCR, Control, Two Tailed Test, One-tailed Test, Sandwich ELISA, Expressing

( A ) Expression levels (in FPKM) of CACLA in various treatment conditions. ( B ) RT-qPCR results show the PTGES mRNA levels in WT cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. Data are shown as mean ± SD from three independent experiments. ( C ) RT-qPCR results show the transcriptional memory of PTGES in WT and RELA KO cells in response to TNF-α. Data are shown as mean ± SD from three independent experiments. One-tailed t -test. ( D ) Locus-specific bisulfite sequencing results of the CALCB promoter for the naïve cells. Filled circles, methylated CpGs; open circles, unmethylated CpGs.

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet: ( A ) Expression levels (in FPKM) of CACLA in various treatment conditions. ( B ) RT-qPCR results show the PTGES mRNA levels in WT cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. Data are shown as mean ± SD from three independent experiments. ( C ) RT-qPCR results show the transcriptional memory of PTGES in WT and RELA KO cells in response to TNF-α. Data are shown as mean ± SD from three independent experiments. One-tailed t -test. ( D ) Locus-specific bisulfite sequencing results of the CALCB promoter for the naïve cells. Filled circles, methylated CpGs; open circles, unmethylated CpGs.

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, One-tailed Test, Methylation Sequencing, Methylation

( A ) Overlap between p65 ChIP-seq peaks and H3K27ac ChIP-seq peaks in cells treated with TNF-α for 12 hr. ( B ) Genome browser view shows p65 occupancy and H3K27ac ChIP-seq signal at the endogenous retrovirus (ERV) region upstream of CALCB gene. ( C ) Locus-specific bisulfite sequencing results of MER11B-left LTR and MER11B-right elements for the cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. ( D ) Locus-specific bisulfite sequencing results show the DNA methylation level of MER11B-left LTR and MER11B-right elements for 0-day and 12-day TNF-α-treated WT, TET1 KO, TET2 KO, TET3 KO, TET TKO, and RELA KO cells. ( E ) Changes of DNA methylation of p65 peaks in 12-day TNF-α-treated cells vs. 0 hr treated cell. Highly methylated and lowly methylated regions are also shown separately. ( F ) DNA methylation around p65 peaks. Colored dots indicate each CpG by relative positions to the binding motif with the highest score. Lowess-smoothed curves were drawn for 0 hr (blue), 12-day TNF-α treatment (green), and the differences between them (red). ( G ) Average methylation level of p65 peaks in WT, RELA KO, and TET2 KO cell with 0 hr or 12-day TNF-α treatment. Only p65 peaks that were initially methylated (methylation level ≥50%) are plotted. ( H ) Left panel shows average methylation level of p65 peaks in WT cells with 0 hr or 12 days TNF-α treatment, categorized by three indicated transcription levels (eRNA at peaks normalized in RPKM). Right panel shows the methylation difference of p65 peaks in RELA KO in comparison to that in WT cells, with or without 12-day TNF-α treatment. Similar to panel G, p65 peaks that were initially methylated (methylation level ≥50%) are used in panel H. Figure 4—source data 1. Related to . Figure 4—source data 2. Related to .

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet: ( A ) Overlap between p65 ChIP-seq peaks and H3K27ac ChIP-seq peaks in cells treated with TNF-α for 12 hr. ( B ) Genome browser view shows p65 occupancy and H3K27ac ChIP-seq signal at the endogenous retrovirus (ERV) region upstream of CALCB gene. ( C ) Locus-specific bisulfite sequencing results of MER11B-left LTR and MER11B-right elements for the cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. ( D ) Locus-specific bisulfite sequencing results show the DNA methylation level of MER11B-left LTR and MER11B-right elements for 0-day and 12-day TNF-α-treated WT, TET1 KO, TET2 KO, TET3 KO, TET TKO, and RELA KO cells. ( E ) Changes of DNA methylation of p65 peaks in 12-day TNF-α-treated cells vs. 0 hr treated cell. Highly methylated and lowly methylated regions are also shown separately. ( F ) DNA methylation around p65 peaks. Colored dots indicate each CpG by relative positions to the binding motif with the highest score. Lowess-smoothed curves were drawn for 0 hr (blue), 12-day TNF-α treatment (green), and the differences between them (red). ( G ) Average methylation level of p65 peaks in WT, RELA KO, and TET2 KO cell with 0 hr or 12-day TNF-α treatment. Only p65 peaks that were initially methylated (methylation level ≥50%) are plotted. ( H ) Left panel shows average methylation level of p65 peaks in WT cells with 0 hr or 12 days TNF-α treatment, categorized by three indicated transcription levels (eRNA at peaks normalized in RPKM). Right panel shows the methylation difference of p65 peaks in RELA KO in comparison to that in WT cells, with or without 12-day TNF-α treatment. Similar to panel G, p65 peaks that were initially methylated (methylation level ≥50%) are used in panel H. Figure 4—source data 1. Related to . Figure 4—source data 2. Related to .

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: ChIP-sequencing, Methylation Sequencing, DNA Methylation Assay, Methylation, Binding Assay, Comparison

( A ) Genome browser view shows p65 occupancy and H3K27ac ChIP-seq signal at the TNF-α- responsive elements of PTGES, LHX2, CCL2, and TNF genes. ( B ) The schematic of κB site (GGGAATTTCC) and its surrounding CpG sites in the MER11B elements adjacent to CALCB . ( C ) Locus-specific bisulfite sequencing results of MER11B-left and MER11B-right elements for the cells with various pretreatments. ( D ) Locus-specific bisulfite sequencing results of MER11B-left and MER11B-right elements for the naïve cells, and the cells that experienced 12 days of TNF-α treatment were cultured in TNF-α-free media for 30 days.

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet: ( A ) Genome browser view shows p65 occupancy and H3K27ac ChIP-seq signal at the TNF-α- responsive elements of PTGES, LHX2, CCL2, and TNF genes. ( B ) The schematic of κB site (GGGAATTTCC) and its surrounding CpG sites in the MER11B elements adjacent to CALCB . ( C ) Locus-specific bisulfite sequencing results of MER11B-left and MER11B-right elements for the cells with various pretreatments. ( D ) Locus-specific bisulfite sequencing results of MER11B-left and MER11B-right elements for the naïve cells, and the cells that experienced 12 days of TNF-α treatment were cultured in TNF-α-free media for 30 days.

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: ChIP-sequencing, Methylation Sequencing, Cell Culture

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet: ( A ) Methylation levels of ten p65 peaks that are associated with the five memory genes. Average values of p65 peaks are shown as circles for 0 hr and 12 days TNF-α-treated WT, RELA KO and TET2 KO cells, respectively. ( B ) H3K27ac ChIP-seq profiles around all p65 peaks (left) and ten peaks that are associated with five memory genes (right). Different colors indicate cells with various treatments. ( C ) Averaged profile of p65 ChIP-seq at p65 peaks grouped by various peak methylation levels. In addition to sequencing depth, read counts located at initially unmethylated p65 peaks (methylation level less than 20%) were used to determine the scale factor for normalization of ChIP-seq signals. Peak-centered 1 kb region are shown. ( D ) Five memory genes, together with EGFP and IL32 , are plotted for their CpG numbers and total demethylation of associated p65 peaks in the flanking 250 bp regions of κB sites. ( E ) Averaged profile of p65 ChIP-seq at methylated p65 peaks grouped by CpG numbers. ( F ) Averaged profile of p65 ChIP-seq at p65 peaks grouped by total methylations in five ranges of quantiles. ( G ) Total methylation level of occupancy-changed p65 peaks that were treated with 0 hr or 12 days TNF-α. Red columns stand for p65 peaks that are relatively higher in the second induction, and green ones stand for p65 peaks that are relatively lower in the second induction (as indicated in ). ( H ) Averaged p65 occupancy at p65 peaks that are categorized into three groups by initial methylation level and total demethylations in 12-day TNF-α treatment vs. 0 hr. The left plot shows initially unmethylated p65 peaks (mCG <20%), the middle one shows initially methylated p65 peaks with the top 25% total demethylation after 12-day TNF-α treatment (higher than the upper quartile, Q3), and the right one shows initially methylated p65 peaks with the lowest 75% total demethylation. The four treatments are shown in indicated colors. ( I ) Averaged ATAC-seq profile at p65 peaks that are categorized into three groups by the same criteria as that used in panel H. ( J ) Averaged profile of H3K27ac ChIP-seq at methylated p65 peaks that are grouped into four ranges of quantiles by total demethylations in 12-day TNF-α treatment vs. 0 hr. The four treatments are shown in indicated colors. ( K ) The number of p65 peaks and their associated genes in designated groups. ( L ) Expression changes of enhancer RNAs at p65 peaks with different degree of demethylation. Paired two-tailed t -test. ( M ) Expression changes of nearest genes of methylated p65 peaks. Paired two-tailed t -test. ( N ) Impact of distance between p65 peaks and TSSs of neighboring genes. Paired two-tailed t -test. Figure 6—source data 1. Related to . Figure 6—source data 2. Related to . Figure 6—source data 3. Related to . Figure 6—source data 4. Related to . Figure 6—source data 5. Related to .

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: Methylation, ChIP-sequencing, Sequencing, Expressing, Two Tailed Test

Aligned reads within p65 peaks were counted and normalized by sequencing depth and peak length of 1 kb, for ChIP-seq in cells treated with 12 hr TNF-α (first induction) and 12 days TNF-α + 10 days + 12 hr TNF-α (second induction), respectively. p65 peaks identified in the two times of induction were combined together. Peaks that are higher or lower than twofold in the second induction are colored in red or green, respectively.

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet: Aligned reads within p65 peaks were counted and normalized by sequencing depth and peak length of 1 kb, for ChIP-seq in cells treated with 12 hr TNF-α (first induction) and 12 days TNF-α + 10 days + 12 hr TNF-α (second induction), respectively. p65 peaks identified in the two times of induction were combined together. Peaks that are higher or lower than twofold in the second induction are colored in red or green, respectively.

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: Sequencing, ChIP-sequencing

Journal: eLife

Article Title: Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness

doi: 10.7554/eLife.61965

Figure Lengend Snippet:

Article Snippet: Samples were incubated with anti-NF-κB p65 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, USA sc-372) overnight at 4°C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software

$Modulation of NF-kB pathway activation by 2J19 and 00123A7 GIB fractions. N9 microglial cells were incubated with 2J19 or 00123A7 for 6 h before LPS-stimulation. ( A ) Immunofluorescence images for the evaluation of NF-κB p65 (red) translocation into the nuclei (cyan). Each capture was chosen as representative of, at least, three independent experiments. ( B ) Kinetics of IκB-α protein levels and ( D ) NF-κB p65 phosphorylation (Ser536) status, both after 0, 15, 30, and 60 min of LPS stimulation. LPS-stimulated cells (control) are denoted by ; cells treated with 2J19 prior to LPS stimulation are denoted by , and cells treated with 00123A7 prior to LPS stimulation are denoted by . ( C ) IκB-α protein levels and ( E ) p65 phosphorylation (Ser536) ratios for each GIB fraction at 0 and 30 min of LPS stimulation (mean ± SEM; n = 3). Statistical differences vs respective timepoint control are denoted as ** p < 0.01; “n.s.” means not statistically different.$

Journal: Antioxidants

Article Title: Bioaccessible Raspberry Extracts Enriched in Ellagitannins and Ellagic Acid Derivatives Have Anti-Neuroinflammatory Properties

doi: 10.3390/antiox9100970

Figure Lengend Snippet: Modulation of NF-kB pathway activation by 2J19 and 00123A7 GIB fractions. N9 microglial cells were incubated with 2J19 or 00123A7 for 6 h before LPS-stimulation. ( A ) Immunofluorescence images for the evaluation of NF-κB p65 (red) translocation into the nuclei (cyan). Each capture was chosen as representative of, at least, three independent experiments. ( B ) Kinetics of IκB-α protein levels and ( D ) NF-κB p65 phosphorylation (Ser536) status, both after 0, 15, 30, and 60 min of LPS stimulation. LPS-stimulated cells (control) are denoted by ; cells treated with 2J19 prior to LPS stimulation are denoted by , and cells treated with 00123A7 prior to LPS stimulation are denoted by . ( C ) IκB-α protein levels and ( E ) p65 phosphorylation (Ser536) ratios for each GIB fraction at 0 and 30 min of LPS stimulation (mean ± SEM; n = 3). Statistical differences vs respective timepoint control are denoted as ** p < 0.01; “n.s.” means not statistically different.

Article Snippet: Then, coverslips were submerged in blocking mixture [1% ( w / v ) BSA, 4% ( v / v ) FBS, 0.4% ( v / v ) Triton, in PBS] for 30 min, before incubation overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) antibody (1:200) (Santa Cruz Biotechnology, Heidelberg, Germany) or rabbit polyclonal anti-NFAT1 antibody (1:200) (Cell Signaling, Danvers).

Techniques: Activation Assay, Incubation, Immunofluorescence, Translocation Assay, Phospho-proteomics, Control

$Scheme of N9 microglia NF-kB, NFAT, MAPK, and IL-10/STAT3 inflammatory pathways modulated by 2J19 GIB fraction. Pre-treatment with 2J19 GIB fraction attenuates N9 microglial pro-inflammatory response to LPS by repressing the NF-κB pathway via inhibition of both p65 phosphorylation and nuclear translocation. MAPK was also inhibited via p38 phosphorylation, as well as NFAT via nuclear translocation upon ATP stimulus. The inhibition of these pathways not only led to a reduction in pro-inflammatory markers CD40, TNF-α, NO, and superoxide, but also led to an increased release of the anti-inflammatory cytokine IL-10. Solid black lines represent evidence-supported effects of 2J19 GIB (poly)phenols; dashed black arrows indicate pathway activation steps supported by literature; solid red arrows represent pro-inflammatory gene expression leading to the indicated molecules; a solid green line indicates the effects of anti-inflammatory gene expression that inhibits the indicated molecules.$

Journal: Antioxidants

Article Title: Bioaccessible Raspberry Extracts Enriched in Ellagitannins and Ellagic Acid Derivatives Have Anti-Neuroinflammatory Properties

doi: 10.3390/antiox9100970

Figure Lengend Snippet: Scheme of N9 microglia NF-kB, NFAT, MAPK, and IL-10/STAT3 inflammatory pathways modulated by 2J19 GIB fraction. Pre-treatment with 2J19 GIB fraction attenuates N9 microglial pro-inflammatory response to LPS by repressing the NF-κB pathway via inhibition of both p65 phosphorylation and nuclear translocation. MAPK was also inhibited via p38 phosphorylation, as well as NFAT via nuclear translocation upon ATP stimulus. The inhibition of these pathways not only led to a reduction in pro-inflammatory markers CD40, TNF-α, NO, and superoxide, but also led to an increased release of the anti-inflammatory cytokine IL-10. Solid black lines represent evidence-supported effects of 2J19 GIB (poly)phenols; dashed black arrows indicate pathway activation steps supported by literature; solid red arrows represent pro-inflammatory gene expression leading to the indicated molecules; a solid green line indicates the effects of anti-inflammatory gene expression that inhibits the indicated molecules.

Techniques: Inhibition, Phospho-proteomics, Translocation Assay, Activation Assay, Gene Expression

Journal: Scientific reports

Article Title: Polyphenols journey through blood-brain barrier towards neuronal protection.

doi: 10.1038/s41598-017-11512-6

Figure Lengend Snippet: Figure 4. Effects on neuroinflammation by Cat-sulf and Pyr-sulf. Pro-inflammatory markers were evaluated, namely (a) TNF-α release, (b) intracellular superoxide production, (c) nitric oxide, and (d) CD40 quantified in N9 microglial cells. Cells were pre-incubated for 6 h with each of the bioavailable (poly)phenol metabolite and then challenged with 300ng/mL of LPS. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to lesion (LPS). (e) Microglial NF-κB p65 translocation into the nucleus after 60 minutes of LPS stimulation. Cells were pre-treated with Cat-sulf or Pyr-sulf for 6 h before LPS-stimulation. NF-κB (red); Nuclei (blue) stained with DAPI. Each capture is representative of at least 3 independent biological replicates. Scale bar: 10 µm. (f–i) Microglial NF-κB p65 phosphorylation ratio and IκBα fold change in protein levels. (f) IkBα protein levels along time after LPS stimulation and (g) after 60 min of LPS stimulation with representative western blots. (h) NF-κB activation profile along time after LPS stimulation looking at NF-κB p65 phosphorylation (ser536) ratio along time after LPS stimulation and (i)after 60 min of LPS stimulation with representative western blots. Cells were pre-treated either with Pyr-sulf or Cat-sulf before LPS stimulation. Control cells (white triangles, solid line), LPS-stimulated cells (black triangles, solid line), cells treated with Cat-sulf prior to LPS stimulation (black circles, dashed line), cells treated with Pyr-sulf prior to LPS stimulation (black squares, dotted line). Statistical differences are denoted as *p < 0.05 and **p < 0.01 relatively to lesion (LPS). Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.

Article Snippet: Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology).

Techniques: Incubation, Translocation Assay, Staining, Phospho-proteomics, Western Blot, Activation Assay, Control

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