mouse monoclonal antibody notch1  (Bio-Rad)

Journal: eLife

Article Title: Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway

doi: 10.7554/eLife.95267

Figure Lengend Snippet:

Article Snippet: Antibody , mouse monoclonal anti-Notch1 , Santa Cruz , #sc-376403 , WB (1:100).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Myeloid Notch1 signaling is closely related to LPS/D-GalN-induced liver injury. ( A ) The expressions of Notch1, Hes1, and Jag1 in the liver tissue of wild-type mice subjected to PBS or LPS (50 μg/kg) combined with D-GalN (700 mg/kg) for 5 hours (n = 5 mice/group). ( B ) Western blot analysis of Notch1 and NICD expression in liver tissue from wild-type mice treated with PBS or LPS/D-GalN injection (n = 5 mice/group). ( C ) Immunofluorescence images of staining with antibodies against F4/80 ( green ) and Notch1 ( red ). Nuclei were labeled with DAPI (blue). n = 5 mice/group. Scale bar, 50 μm. ( D ) Hepatic macrophages were isolated from mice after PBS or LPS/D-GalN injection. Notch1 and NICD expression was examined by immunoblotting. The correlation between serum ALT ( E ) or AST levels ( F ) and Notch1 expression in hepatic macrophages after LPS/D-GalN injection (n = 15 mice). The correlation coefficient was calculated by the Pearson correlation test. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Western Blot, Expressing, Injection, Immunofluorescence, Staining, Labeling, Isolation, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Myeloid-specific deletion of Notch1 alleviates LPS/D-GalN-induced liver inflammation. ( A ) The NICD expression was detected by Western blot assay in hepatic macrophages from the Notch1 FL/FL and Notch1 M-KO livers. ( B ) The representative gross appearance of the collected livers and histologic staining (H&E) of liver sections from Notch1 FL/FL and Notch1 M-KO mice after PBS or LPS/D-GalN injection (n = 5 mice/group). Scale bars, 100 μm. ( C ) Liver function in serum samples was evaluated by serum ALT and AST levels (IU/L) (n = 5 samples/group). ( D ) Immunohistochemistry staining and quantification of Ly6G + neutrophils and F4/80 + macrophages in liver sections (n = 5 mice/group). Scale bars, 40 μm. ( E ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of proinflammatory cytokines ( Tnf-α , Il-6 , and Il-1β ), chemokines ( Mcp-1 and Cxcl-1 ), and anti-inflammatory cytokines ( Il-10 and Tgf-β ) in liver tissues (n = 5 samples/group). ( F ) Kaplan-Meier survival curve comparing percent survival of Notch1 FL/FL and Notch1 M-KO mice treated with LPS (50 μg/kg) and D-GalN (700 mg/kg) (n = 9–11 mice/group). Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Expressing, Western Blot, Staining, Injection, Immunohistochemistry, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Myeloid-specific deletion of Notch1 alleviates LPS/D-GalN-induced hepatocellular apoptosis. ( A ) Representative TUNEL staining images and quantification of TUNEL + cells in liver sections from the Notch1 FL/FL and Notch1 M-KO mice treated with PBS or LPS/D-GalN injection (n = 5 mice/group). Scale bars, 20 μm. ( B ) Immunohistochemistry staining and quantification of cleaved caspase-3 (C-caspase-3) positive cells in liver sections (n = 5 mice/group). Scale bars, 40 μm. ELISA analysis of serum TNF-α ( C ) and HMGB1 ( D ) levels in the Notch1 FL/FL and Notch1 M-KO mice (n = 5 samples/group). ( E ) Western blot analysis and relative density ratio of p-ASK1, ASK1, p-p38, p38, C-caspase-3, caspase-3, Bcl-xL, and Bax in the Notch1 FL/FL and Notch1 M-KO livers. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: TUNEL Assay, Staining, Injection, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Myeloid Notch1 deficiency depresses YAP signaling and reprograms macrophage polarization. ( A ) Flow cytometry analysis of liver macrophages isolated from Notch1 FL/FL and Notch1 M-KO mice (n = 5 mice/group). ( B ) The Arg1 and iNOS expression were detected by Western blot assay in liver macrophages from Notch1 FL/FL and Notch1 M-KO mice treated with PBS or LPS/D-GalN injection. ( C ) Quantitative reverse-transcriptase polymerase chain reaction analysis of Yap , Ctgf , and Cyr61 in liver macrophages (n = 5 samples/group). ( D ) The expression of YAP in liver macrophages from Notch1 M-KO and Notch1 FL/FL mice, as measured by Western blot analysis. ( E ) The correlation between serum ALT levels and Yap mRNA expression in liver macrophages after LPS/D-GalN injection (n = 15 mice). The correlation coefficient was calculated by the Pearson correlation test. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Flow Cytometry, Isolation, Expressing, Western Blot, Injection, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: YAP is required for Notch1-mediated macrophage polarization and liver inflammation. BMDMs from Notch1 FL/FL and Notch1 M-KO mice were transfected with lentivirus expressing YAP (Lv-YAP) or GFP control (Lv-GFP) and adoptively transferred into Notch1 FL/FL and Notch1 M-KO mice, respectively. ( A ) Representative H&E staining of liver sections (n = 5 mice/group). Scale bars, 100 μm. ( B ) The serum ALT and AST levels from the indicated groups. ( C, D ) Representative immunohistochemistry staining and quantification of F4/80 + macrophages and Ly6G + neutrophils in liver sections (n = 5 mice/group). Scale bars, 40 μm. ( E ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of proinflammatory cytokines ( Tnf-α , Il-6 , and Il-1β ), chemokines ( Mcp-1 and Cxcl-1 ), and anti-inflammatory cytokines ( Il-10 and Tgf-β ) in liver tissues (n = 5 samples/group). ( F ) Flow cytometry analysis of liver macrophages isolated from Notch1 FL/FL and Notch1 M-KO mice (n = 5 mice/group). ( G ) The expression of Arg1 and iNOS in liver macrophages from Notch1 FL/FL and Notch1 M-KO mice, as measured by Western blot analysis. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Transfection, Expressing, Control, Staining, Immunohistochemistry, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Isolation, Western Blot, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Notch1 activation upregulates YAP signaling in macrophages. ( A ) BMDMs were isolated from Notch1 M-KO and Notch1 FL/FL mice and stimulated with LPS (100 ng/mL)/IFN-γ (10 ng/mL) for 12 hours. Western blot analysis of NICD and Arg1 expression in BMDMs. ( B-D ) BMDMs were transfected with pEF-Flag-NICD plasmid or control vector. After 48 hours, cells were supplemented with LPS (100 ng/mL)/IFN-γ (10 ng/mL) for an additional 12 hours. ( B ) Western blot analysis of NICD and Arg1 expression in indicated BMDMs. ( C ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of Hes1 , Yap , Ctgf , and Cyr61 in indicated BMDMs. ( D ) Western blot analysis of Hes1 and YAP in indicated BMDMs. ( E ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of Hes1 , Yap , Ctgf , and Cyr61 in BMDMs from Notch1 M-KO and Notch1 FL/FL mice. ( F ) Western blot analysis of Hes1 and YAP expression in BMDMs from Notch1 M-KO and Notch1 FL/FL mice. ( G ) BMDMs were cotransfected with YAP-luciferase and pEF-Flag-NICD vectors, and the luciferase activity was measured after 48 hours. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Activation Assay, Isolation, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Polymerase Chain Reaction, Luciferase, Activity Assay, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: YAP activation upregulates JAG1 in macrophages and contributes to Notch1-mediated macrophage polarization in vitro. ( A, B ) BMDMs were transfected with YAP-siRNA or NS-siRNA and stimulated with LPS (100 ng/mL)/IFN-γ (10 ng/mL) for 12 hours. ( A ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of Ctgf , JAG1 , Notch1 , and Hes1 . ( B ) Western blot analysis of YAP and JAG1 expression. ( C ) The expression of YAP and JAG1 in BMDMs transfected with pCMV-Flag-YAP plasmid or control vector, as measured by Western blot analysis. ( D ) BMDMs were cotransfected with JAG1-luciferase and pCMV-Flag-YAP vectors, and the luciferase activity was measured after 48 hours. ( E, F ) Notch1 M-KO BMDMs were transfected with pCMV-Flag-YAP plasmid or control vector followed by LPS/IFN-γ stimulation. ( E ) Western blot analysis of Arg1 and iNOS expression in BMDMs. ( F ) Representative immunofluorescence staining of iNOS in BMDMs. Scale bars, 200 μm. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Activation Assay, In Vitro, Transfection, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Immunofluorescence, Staining, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Inhibition of YAP in macrophages attenuates LPS/D-GalN-induced liver injury. Notch1 FL/FL mice were injected by the tail vein with YAP-siRNA or NS control siRNA mixed with mannose-conjugated polymers at 6 hours before LPS/D-GalN treatment (n = 5 mice/group). ( A ) Representative H&E staining of liver sections. ( B ) Serum ALT and AST levels (U/L). ( C, D ) Immunohistochemical staining and quantification of Ly6G + neutrophils and F4/80 + macrophages in liver sections. Scale bars, 40 μm. ( E ) Representative TUNEL-staining images and quantification of TUNEL + cells in liver sections. Scale bars, 20 μm. ( F ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of proinflammatory cytokines ( Tnf-α , Il-6 , and Il-1β ), chemokines ( Mcp-1 and Cxcl-1 ), and anti-inflammatory cytokines ( Il-10 and Tgf-β ) in liver tissues. Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Inhibition, Injection, Control, Staining, Immunohistochemical staining, TUNEL Assay, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: Dual inhibition of Notch1-YAP circuit attenuates LPS/D-GalN-induced liver inflammation and damage. Notch1 M-KO mice were injected by the tail vein with YAP-siRNA or NS control siRNA mixed with mannose-conjugated polymers at 6 hours before LPS/D-GalN treatment. ( A ) Representative H&E staining of liver tissue. ( B ) The hepatocellular function was evaluated by serum ALT and AST levels (U/L). ( C, D ) Immunohistochemical staining and quantification of Ly6G + neutrophils and F4/80 + macrophages in liver sections (n = 5 mice/group). Scale bars, 40 μm. ( E ) Quantitative reverse-transcriptase polymerase chain reaction–assisted detection of proinflammatory cytokines ( Tnf-α , Il-6 , and Il-1β ), chemokines ( Mcp-1 and Cxcl-1 ), and anti-inflammatory cytokines ( Il-10 and Tgf-β ) in liver tissues (n = 5 samples/group). ( F ) Representative TUNEL-staining images and quantification of TUNEL + cells in liver sections (n = 5 mice/group). Scale bars, 20 μm. ( G ) Flow cytometry analysis of liver macrophages isolated from Notch1 M-KO mice with YAP-siRNA or NS-siRNA treatment (n = 5 mice/group). Data are presented as the mean ± standard deviation. ∗ P < .05, ∗∗ P < .01.

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques: Inhibition, Injection, Control, Staining, Immunohistochemical staining, Reverse Transcription, Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Isolation, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

doi: 10.1016/j.jcmgh.2023.01.002

Figure Lengend Snippet: List of Primers Used in This Study

Article Snippet: F4/80 and Notch1 double-positive macrophages were identified by rat antimouse F4/80 (Bio-Rad, Hercules, CA) and mouse monoclonal antibody Notch1 (Santa Cruz Biotechnology) followed by incubating with secondary Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (H+L) or Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch), respectively.

Techniques:

Journal: PLoS ONE

Article Title: Regulation of PTEN/Akt Pathway Enhances Cardiomyogenesis and Attenuates Adverse Left Ventricular Remodeling following Thymosin β4 Overexpressing Embryonic Stem Cell Transplantation in the Infarcted Heart

doi: 10.1371/journal.pone.0075580

Figure Lengend Snippet: A . Representative photomicrographs of immunostained tissue sections showing S-actin in red (a, e, i, m), RFP in green (b, f, j, n), dapi in blue (c, g, k, o) and merged images in pink (d, h, l, p). Scale = 50µm. B . Quantitative analysis shows a significant increase in cardiac myocyte differentiation in the Tβ4-ESC group. *p< vs. MI, #p<0.05 vs. RFP-ESCs. C . Representative photomicrographs showing S-actin in red (a), RFP in green (b), Notch1 in purple (c), dapi in blue (d), merged images in pink (e), and at high resolution (f). Scale=100µm. D . Histogram shows expression levels of Notch-1 in all groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs.

Article Snippet: For Notch-1 staining, sections were incubated with Notch-1 mouse monoclonal antibody (1:40, Abcam) using the M.O.M. kit (Vector Laboratories) in a humidified chamber.

Techniques: Expressing