Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 2: Overexpression of UGDH in the AD LNCaP background desensitizes AR-mediated gene expression and reduces glucuronidation precursors. Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP AD background. Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells were harvested for analysis. AR-dependent genes PSA (A) and UGT2B17 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by LC-MS. In panels A–C, mean ± SEM is plotted for triplicate measurements. In panel D, mean ± SD is plotted for quadruplicate measurements. Statistical significance is indicated as: (a) p < 0.05 relative to VC1 at 0 nM DHT. (b) p < 0.05 relative to VC2 at 0 nM DHT. (c) p < 0.05 comparing OE1 to both VC1 and VC2 at the indicated [DHT]. (d) p < 0.05 comparing OE2 to both VC1 and VC2 at the indicated [DHT].

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Over Expression, Gene Expression, Plasmid Preparation, Control, Concentration Assay, Functional Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 3: Overexpression of UGDH in the CR LNCaP background further suppresses AR-mediated expression of glucuronidation genes and reduces nucleotide sugar pools without impacting proteoglycan production. Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP CR background. Equal cell numbers were seeded 48 hours in androgen replete media followed by harvest of cells for analysis of gene expression (A and B) and UDP-sugars (D). For panel (C), equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells and media were harvested for analysis. AR-dependent genes UGT2B17 (A) and FoxA1 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by mass spectrometry. Mean ± SEM is plotted for triplicate technical measurements; *p < 0.05 for OE1 and OE2 relative to VC1 and VC2.

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Gene Expression, Concentration Assay, Functional Assay, Mass Spectrometry

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 5: Loss of UGDH promotes AR-dependent gene expression and reduces proteoglycan production while sustaining glucuronide output. LNCaP AD and CR cells were selected for stable expression of a non-targeting vector control (VC1 and VC2) or a UGDH shRNA knockdown construct (KD1 and KD2). Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing 10 nM DHT or DMSO (vehicle, 0 nM). After an additional 48 hours, cells were harvested for analysis by western blot and mass spectrometry. (A) AR-dependent genes PSA (panels a and d) and UGT2B17 (panels b and e) were analyzed by WB in whole cell lysates; functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates (panels c and f). Panels (a–c) depict expression data in the AD background and panels (d–f) illustrate data from the CR background. Mean ± SEM is plotted. Statistical significance is indicated on the plots as: (a) p < 0.05 for that clone, comparing 0 vs 10 nM DHT; (b) p < 0.05 relative to VC1 and VC2, 10 nM DHT; (c) p < 0.05 relative to VC1 and VC2, 0 nM DHT. (B) UDP-sugar pools were measured in cell lysates by LC-MS for both the AD (upper) and CR (lower) backgrounds as indicated. Mean ± SD is plotted. Statistical significance is indicated as: (a) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 0 nM DHT. (b) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 10 nM DHT. (c) p < 0.05 for that clone, comparing 0 and 10 nM DHT.

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Gene Expression, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Construct, Western Blot, Mass Spectrometry, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 4 (a) Selected gene expressions regarding epithelial mesenchymal markers, differentiation makers and regulators from RNA- sequencing data. (b and c) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day4 after siRNA. *p < 0.05. Data presented are from one of two independent experiments with similar results. (d and e) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR. *p < 0.05. Data presented are from one of two independent experiments with similar results. (f) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with siRNA and 3D organoid matrigel. *p < 0.05. Data presented are from one of two independent experiments with similar results. (g) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Transfection

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 5 TINCR binds to STAU1 protein and controls critical regulators of differentiation. (a) Bar graphs show percentage of TINCR in the cytoplasm (black) and nucleus (white). NEAT1 serves as a positive control for nucleus enriched RNA and GAPDH serves as a positive control for cytoplasmic RNA. Data presented are from two independent experiments. (b) RIP experiments were performed using isotype IgG and STAU1 antibody to immunoprecipitated STAU1 protein/mRNAs complexes in total-cell extracts of NHBECs, and relative enrichment was determined as RNA associated with STAU1 IP relative to an input control. Relative ARF1 enrichment served as a positive control and NEAT1 as a negative control as NEAT1 does not interact with STAU1. Data presented are from two independent experiments. (c) Relative mRNA enrichment of STAU1 antibody in total-cell extracts of NHBECs transfected with siSCR or siTICNR. Data presented are from two independent experiments. (d and e) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU1. NHBECs were seeded on 6 well plate at 2 × 105 density and analyzed at day4 after transfection of siRNA reagents. *p < 0.05. (f and g) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU after transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR for 4 h. NHBECs were seeded on 24 well plate at 1 × 105 density and analyzed at day4 after transfection of siRNA reagents

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: Positive Control, Immunoprecipitation, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Characterization of Notch1 expression in sporadic AD brain tissue. a – b ’) Immunohistochemical analysis on hippocampus ( a – a ’) and entorhinal cortex ( b – b ’) shows an aberrant Notch1 deposition in the brain parenchyma. Moreover, in AD patients, Notch1 labeling appears fainter in the cell bodies and processes of cortical and hippocampal neurons as compared to healthy controls ( a – b ). a ’- b ’) Notch1 positive plaques with different conformation either core plaques (upper captions) or diffuse distribution (bottom captions). c – d ) Box plots summarizing the quantification of Notch1 positive pixels occupying the soma of ( c ) hippocampal and ( d ) cortical neurons of AD and healthy control patients ( p <0.001 for both regions). e ) Double immunofluorescence analysis on the dorsal hippocampus indicates that, in AD tissue, Notch1 is deposited in A β 42 positive plaques and is decreased in neuronal soma as compared to healthy controls. Nuclei have been counterstained using DAPI. f ) Bar graph showing the counting of plaques/100 μ m 2 , in the hippocampus, positive for Notch1, A β 42 and double positive. g ) Bar graph showing the counting of plaques per 100 μ m 2 , in the entorhinal cortex, positive for Notch1, A β 42 and double positive. *= p <0.05, **= p <0.01. Error bars are SEM. Scale bars are 100 μ m in ( a ) and ( e ), and 25 μ m in ( a ’) and ( b ’)

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Expressing, Immunohistochemical staining, Labeling, Immunofluorescence

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 intracellular and extracellular domains have distinct patterns in AD brains. a – a ”) Fluorescent immunolabeling indicates that the Notch1 extracellular domain is abundantly expressed in the processes and is co-distributed with the Notch1 cytoplasmic fragment on the cell body of healthy neurons ( a ). In degenerating neurons (arrowhead in a ’), the extracellular domain of Notch1 is strongly expressed in the soma and process, whereas the cytoplasmic portion of the receptor is condensed into the nucleus (arrowhead in a ’). In addition, the two domains of Notch1 co-label some A β 42 stained plaques (white arrows in a ’ and insert in a ") but not all (light blue arrows in a ’). Notably, the Notch1 extracellular portion is strongly present in the amyloid fibrils, while the intracellular component is present at a lower level ( a "). Scale bars in all panels are 40 μ m

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Immunolabeling, Staining

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 is expressed in A β 42 positive plaques invaded by activated microglia and astroglia. a – a” ) Fluorescent immunolabeling reveals that in AD patients, A β 42 and Notch1 are present in core plaques (arrows in a’ and magnified insert) and fibrillary-like plaques ( a” ) with abundant activated microglia, CD68 positive. In ( a’ ) a magnified insert shows a microglia (blue) ensheathed in an amyloid fibrillary Notch1 positive plaque. In the healthy controls ( a ), A β 42 and CD68 immunoreactivity is significantly lower and Notch1 labels homogeneously the neuronal soma (insert). b – b” ) Representative fluorescent immunolabeling shows that in AD patients, core plaques and fibrillary structures positive for Notch1 and A β 42 are invaded by GFAP positive astroglia. GFAP immunoreactivity is significantly lower in healthy controls ( b ) as compared to the AD samples b’ - b” ). Scale bars in all panels are 40 μ m

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Immunolabeling

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 distribution in Thioflavin T positive plaques and fibrillary aggregates. a – a” ) Double immunofluorescence indicates the co-expression of Notch1 and Thioflavin T stained fibrils in AD brains ( a’ - a" ) as compared to the age matched control ( a ). In non-demented patients, Notch1 is homogeneously distributed in neurons and Thioflavin T signal is not present. b and c ) Bar graphs showing the counting, of single or double labeled plaques for Notch1 and Thioflavin T in the hippocampus ( b ) and in the entorhinal cortex ( c ). d - d’ ) Fluorescence immunostaining of the entorhinal cortex for Notch1, APP and Thioflavin T shows that Notch1 is mainly localized with Thioflavin T deposits (arrows in d’ and insert) and to a lower extent with APP in AD brains. Expression of Notch1 and APP is critically altered in AD brains as compared to healthy control tissue ( d ). **= p <0.01. Error bars are SEM. Scale bars are 50 μ m in all images

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Immunofluorescence, Expressing, Staining, Labeling, Fluorescence, Immunostaining

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Intracellular depositions of Notch1 and p-Tau are increased in AD degenerating neurons. a – a’ ) Double immunostaining showing the enhanced number of damaged neurons labeled with p-Tau and partially with Notch1 in AD and healthy control cortical tissue. b ) Triple staining showing the co-expression of Notch1 and p-Tau in neurofilament positive degenerating cortical neurons (insert). c – d ) Bar graphs indicating the counting of degenerating neurons positive for p-Tau or double positive for p-Tau and Notch1, either in the hippocampus ( c ) or in the entorhinal cortex ( d ). *= p <0.05, **= p <0.01, Error bars are SEM. Scale bar in ( a’ ) is 100 μ m and in ( b ) 40 μ m

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Double Immunostaining, Labeling, Staining, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 colocalizes with p-Tau in fibrillary plaques. a – a ’) In the entorhinal cortex of demented patients, Notch1 costaining with p-Tau surrounds Thioflavin T labeled plaques (arrows in a ’), whereas in the healthy control ( a ) there is no presence of aberrant aggregates. b – b ’) In the AD cortical tissue, Notch1 is co-expressed with p-Tau in NF-200 positive neurofibrillary filaments (arrows in b ’), whereas there is no apparent colocalization in the healthy control tissue ( b ). c – d ) Bar graphs indicating the amount of plaques either in the hippocampus ( c ) or in the entorhinal cortex ( d ) positive for Notch1, p-Tau and double stained. *= p <0.05, **= p <0.01, ***= p <0.001. Error bars are SEM. In all panels scale bars are 40 μ m

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Labeling, Staining

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 levels in the entorhinal cortex of AD patients. a ) Representative Western Blot analysis on whole cell lysate indicates no difference in Notch1 levels, represented by the extracellular and the intracellular component, between healthy controls and AD patients. In contrast, p-Tau is increased in AD lysates. b – e ) Western blot on synaptosomal fractions shows the unchanged expression of Notch1 in the synaptic (P2) component ( p =0.2) of AD samples as compared to the controls ( b and c ). On the other hand, in the cytoplasmic compartment (S2) Notch1 levels are increased ( p =0.007) in AD samples as compared to the controls ( d and e ). In addition, in both fractions, the levels of p-Tau are augmented in AD samples, (P2, p =0.02; S2, p =0.002) as compared to the age matched controls ( c and e ). f – g ) Immunoblotting ( f ) and bar graph ( g ) on nuclear fraction indicates no difference in Notch1 expression between AD and healthy controls ( p =0.25). *= p <0.05, **= p <0.01. Error bars are SEM

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 signaling in AD patients. a ) Immunohistochemistry using an antibody specific for NICD1 shows that in AD cortices the activation of Notch1 in pyramidal neurons is lower as compared to the healthy controls (inserts). b – c ) Box plots summarizing the NICD signal shows a significant difference in immunoreactivity in ( b ) hippocampal and ( c ) cortical neurons between AD and healthy CTLs. d ) Bar graph showing the fold change in transcript levels of Notch1 and some target genes, such as Hes1 , Hey1 and BDNF . Only the mRNA of Notch1 is near to significance (0 = p =0.06). Error bars are SEM. Scale bar in ( a ) is 50 μ m

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Immunohistochemistry, Activation Assay

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Notch1 fragments are less represented in the CSF from AD patients. a ) Immunoblotting on CSF reveals that the full length (FL) of Notch1 is decreased in AD samples as compared to healthy controls. Moreover, other fragments of the protein around 55 and 28 KDa appear decreased in AD samples. b ) Bar graph showing the pattern of expression of Notch1 FL and the smaller fragments in AD samples as compared to healthy controls. c ) Immunoblotting on CSF showing the diminished level of the extracellular portion of Notch1 in AD samples as compared to age matched controls. Also with this antibody we detected fragments, either around 55 or 28 KDa, which are reduced in AD CSF. d ) Bar graph showing the reduction of Notch1 extracellular domain and the smaller truncations of the receptor in AD samples. *= p <0.05, **= p <0.01, ***= p <0.001. Error bars are SEM

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Notch1 hallmarks fibrillary depositions in sporadic Alzheimer’s disease

doi: 10.1186/s40478-016-0327-2

Figure Lengend Snippet: Altered Notch1 expression and deposition in the liver of AD patients. a - b ) Immunofluorescence on liver sections. a ) Notch1 and A β 42 positive aggregates (arrows) in the hepatic parenchyma of AD patients, but not in controls. Moreover, Notch1 is highly expressed in the cytosol of several hepatocytes (arrowsheads). b ) Antibodies specific for the extracellular and intracellular portions of Notch1 strongly label hepatocytes in the AD liver as compared to the healthy control section. c ) Box plot summarizing the mean grey value intensity of the Notch1 intracellular signal in AD and CTL hepatocytes. d ) Box plot summarizing the mean grey value intensity of the Notch1 extracellular signal in AD and CTL hepatocytes. The scale bars are in ( a ) and ( b ) 50 μ m

Article Snippet: The primary antibodies utilized for the western blot analysis on entorhinal cortex sections and CSF were goat anti-Notch1, 1:500 (sc-6014; Santa Cruz Biotechnology, USA), goat anti-Notch1 extracellular portion, 1:500 (sc-23299; Santa Cruz Biotechnology, USA), rabbit anti-Notch1 cytoplasmic domain, 1:500 (cat. no. 07-220; Millipore, USA), rabbit anti-phosphorylated Tau, 1:1000 (phospho T205/ab4841; Abcam, UK), mouse anti-PSD95, 1:1000 (sc-32290; Santa Cruz Biotechnology, USA), rabbit anti-NeuN, 1:1000 (ab177487; Abcam, UK), mouse anti- β actin, 1:2000 (sc-81178; Santa Cruz Biotechnology, USA), mouse anti-Gapdh, 1:8000 (sc-365062; Santa Cruz Biotechnology, USA).

Techniques: Expressing, Immunofluorescence

Journal: International journal of molecular sciences

Article Title: Nrf2 Transcriptional Activity Governs Intestine Development.

doi: 10.3390/ijms23116175

Figure Lengend Snippet: Figure 5. Notch1 is reduced in the Nrf2 tKO embryos at the latest stages of development. (A) Notch1 expression in the female and male fetuses at selected gestation days. (B) Quantification of Notch1 in the hindgut of the Nrf2 WT and Nrf2 tKO fetuses. N = 3–11 fetuses for the Nrf2 WT and Nrf2 tKO mice. Mean ± SEM. Two-way ANOVA. ** p < 0.01. Representative images, magnification 200×, scale bar 50 µm.

Article Snippet: After washing in PBS, the samples were incubated overnight (4 ◦C) with mouse anti-Ki67 monoclonal IgG antibodies (dilution 1:500; Abcam), rabbit antiNrf2 IgG polyclonal antibodies (dilution 1:200; Proteintech), or rabbit anti-Notch1 IgG monoclonal antibodies (dilution 1:250; Cell Signaling) diluted in 3% GS in PBS with 0.05% Tween-20.

Techniques: Expressing