Journal: Neoplasia (New York, N.Y.)

Article Title: Downregulation of Notch Signaling in Kras-Induced Gastric Metaplasia

doi: 10.1016/j.neo.2019.06.003

Figure Lengend Snippet: Differential expression of Notch receptors in the forestomach and glandular stomach lesions in Kras LSL-G12D/+ ;Pdx1-cre mice. (A-C) Immunostaining of Notch1 in normal squamous epithelium (A) and squamous cell carcinoma (B) of the forestomach, and metaplastic lineages of the corpus (C). (D-F) Immunostaining of Notch2 in normal squamous epithelium (D), squamous cell carcinoma (E), and metaplastic lineages of the corpus (F). (G-I) Immunostaining of Notch3 in normal squamous epithelium (G), squamous cell carcinoma (H), and metaplastic lineages of the corpus (I). (J-L) Immunostaining of Notch4 in normal squamous epithelium (J), squamous cell carcinoma (K), and corpus metaplastic lineages (L). Scale bars: 50 μm.

Article Snippet: Primary antibodies used for immunostaining were: GFP (Invitrogen, A11122, 1:200), Ki67 (Abcam, ab16667, 1:100), Mist1 (Santa Cruz, sc-80984, 1:100), Jagged1 (Santa Cruz, sc-6011, 1:100), Notch1 (Cell Signaling, No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, 55114-1-AP, 1:100), Notch4 (Millipore, 09-089, 1:100), Cytokeratin 19 (Abcam, ab52625, 1:200), Muc5AC (Santa Cruz, sc-21701, 1:100), Clusterin (Santa Cruz, sc-6420, 1:100), and TFF3 (ProteinTech, 23277-1-AP, 1:100).

Techniques: Quantitative Proteomics, Immunostaining

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 1. Notch1-shRNA suppresses L02/HBx cell proliferation in vitro. (A) Identification of the effective shRNA targeting the Notch1 gene. The relative mRNA and protein levels of Notch1 were assessed by qRT-PCR and western blotting in L02/HBx cells 48 h after transient transfection with Notch1-shRNA1, Notch1-shRNA2 or negative control-shRNA (NC), respectively. (B) The components of the Notch1 signaling pathway were downregulated in the L02/ HBx-Notch1 shRNA2 cells. The mRNA and protein expression levels of Notch1 and Hes1 were assessed by qRT-PCR and western blotting, respectively. Actin was used as a loading control for both quantitative RT-PCR and western blotting. (C) CCK-8 assay and (D) colony formation assay of L02/HBx cells stably transfected with control or Notch1 shRNA2. Data are shown as the mean ± SEM from at least three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, In Vitro, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Expressing, Control, CCK-8 Assay, Colony Assay, Stable Transfection

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 2. Notch1-shRNAs suppresses L02/HBx cell proliferation in vivo. (A) The growth curve of the tumors derived from L02/HBx cells pretreated with Notch1-shRNA or control-shRNA in nude mice. Data represent means ± SEM of six samples. *P<0.05, **P<0.01. (B) Images of representative mice and dis sected tumors from nude mice. (C) Tumor tissues from nude mice inoculated with NC or Notch1-shRNA cells were stained with hematoxylin and eosin (H&E). Original magnification, x200. (D) Immunohistochemistry of Notch1 expression in tumor tissues from nude mice. Original magnification, x400.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: In Vivo, Derivative Assay, shRNA, Control, Staining, Immunohistochemistry, Expressing

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 3. Notch1-shRNA induces cell cycle arrest via the CyclinD1/CDK4 pathway in L02/HBx cells. (A) Notch1-shRNA induced cell cycle arrest in L02/ HBx cells. Cell cycle distribution was examined by flow cytometry after staining with PI. Results are visualized as a representative experiment or means ± SEM of three experiments. (B) Notch1-shRNA downregulates the CyclinD1/CDK4 pathway in L02/HBx cells. The expression levels of cell cycle regulatory genes CyclinD1, CDK4, E2F1, Rb, p21 and CyclinE1 were analyzed by qRT-PCR and western blotting. Data represent the mean ± SEM from three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, Cytometry, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 4. Notch1-shRNA increases cell apoptosis via the caspase-9-caspase-3 pathway in L02/HBx cells. (A) Notch1-shRNA increased cell apoptosis in L02/ HBx cells. Analysis of apoptosis was performed using PE Annexin V and 7-AAD staining by FACS analysis. Results are visualized as a representative experi ment or means ± SEM of three experiments. (B) Notch1-shRNA upregulated the caspase-9-caspase-3 pathway in L02/HBx cells. The expression of caspase-9, -8 and -3 was analyzed by qRT-PCR and western blotting. Data are shown as the mean ± SEM of three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Food Science & Nutrition

Article Title: Vitamin D ameliorates asthma‐induced lung injury by regulating HIF‐1α/Notch1 signaling during autophagy

doi: 10.1002/fsn3.2880

Figure Lengend Snippet: The primer sequences

Article Snippet: G1285 and G1243, respectively; Solarbio Life Science); interleukin (IL)‐6, IL‐10, and IL‐17 enzyme‐linked immunoassay (ELISA) kits; (Shanghai Jingtian Biotechnology Co., Ltd.); TUNEL kit (cat. no. KGA703; KeyGen Biotech); rabbit antimouse primary antibody LC 3B (cat. no. ab225383; Abcam), Beclin1 (cat. no. ab207612; Abcam), P62 (cat. no. ab91526; Abcam), HIF‐1α (cat. no. ab51608; Abcam), and Notch1 (cat. no. ab276343; Abcam); GAPDH (cat. no. ab181602; Abcam); goat antirabbit secondary antibody (batch No. SA00001‐2; Proteintech); Trizol reagent (batch No. 15596026; Ambion, Inc.); reverse transcription kit (batch no.: K1622; Thermo Scientific); real‐time fluorescent quantitative polymerase chain reaction (real‐time qPCR) kit (batch No. PA0025; Shanghai Jinan Biotechnology Co., Ltd.).

Techniques:

Journal: Food Science & Nutrition

Article Title: Vitamin D ameliorates asthma‐induced lung injury by regulating HIF‐1α/Notch1 signaling during autophagy

doi: 10.1002/fsn3.2880

Figure Lengend Snippet: HIF‐1α (hypoxia‐inducible factor 1α) and Notch1 (neurogenic locus notch homolog protein 1) expression by western blotting analysis. Normal: Mice treated with normal saline; Model: asthmatic model mice treated with normal saline; LD: asthmatic model mice treated with low‐dose Vit D (100 IU/kg/day); MD: asthmatic model mice treated with middle‐dose Vit D (500 IU/kg/day); HD: asthmatic model mice treated with high‐dose Vit D (1000 IU/kg/day). *** p < .001, compared with Normal; # p < .05, ## p < .01, ### p < .001, compared with Model; $ p < .05, $$ p < .01, compared with LD group; & p < .05, compared with MD group

Article Snippet: G1285 and G1243, respectively; Solarbio Life Science); interleukin (IL)‐6, IL‐10, and IL‐17 enzyme‐linked immunoassay (ELISA) kits; (Shanghai Jingtian Biotechnology Co., Ltd.); TUNEL kit (cat. no. KGA703; KeyGen Biotech); rabbit antimouse primary antibody LC 3B (cat. no. ab225383; Abcam), Beclin1 (cat. no. ab207612; Abcam), P62 (cat. no. ab91526; Abcam), HIF‐1α (cat. no. ab51608; Abcam), and Notch1 (cat. no. ab276343; Abcam); GAPDH (cat. no. ab181602; Abcam); goat antirabbit secondary antibody (batch No. SA00001‐2; Proteintech); Trizol reagent (batch No. 15596026; Ambion, Inc.); reverse transcription kit (batch no.: K1622; Thermo Scientific); real‐time fluorescent quantitative polymerase chain reaction (real‐time qPCR) kit (batch No. PA0025; Shanghai Jinan Biotechnology Co., Ltd.).

Techniques: Expressing, Western Blot, Saline

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Notch was found up-regulated after azelaic acid (AZA) treatment. (A) 120 differential expressed proteins associated in 52 functions after AZA treatment by protein mass spectrometry analysis in the heat map. (B) GO annotation with 528 differential expressed proteins. (C, D) THP-1, Molm-13, NK, and T cells were treated with 10 µM AZA for 24 h, the expression level of ICN1 and ICN2 was validated by western blot. (E) The expression of Notch1 and Notch2 in mouse spleen measured by ICH. IHC, immunohistochemistry.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Mass Spectrometry, Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Azelaic acid (AZA) exerts anti-leukemic effect by activating the Notch signaling pathway. (A) Notch responsive elements were transfected into 293T cells after 24 h. Cells were then treated with 10 µM AZA, 10 µM RO4929097, and combination for 24 h, the Notch signaling reporter assay was measured by dual luciferase reporter activity. (B) Validation of the RNA expression of Notch1 and Notch2, the downstream target genes HES1 and HEY1 in Molm-13 and THP-1 cells by qPCR. (C) The protein expression level of ICN1, ICN2, HEY1, and HES1 in acute myeloid leukemia (AML) cell lines after treatment of AZA and RO4929097 and their detection by western blot. ImageJ was used for the densitometric analysis. Data represent means ± SD. (D) Molm-13 cells were pretreated with 10 µM RO4939097 for 24 h, and then treated with 10 µM AZA for another 24 h. Cells were collected for apoptosis analysis. (E) NK cells and T cells were pretreated with 10 µM AZA, 10 µM RO4929097, and combination for 24h before co-culture with THP-1 cell and Molm-13 cells at an E:T ratio of 5:1. The cytotoxicity of NK and T was determined by detecting the LDH release rate. (F) NK and T cells were pre-treated with AZA and RO4929097, then co-cultured with THP-1 cell at an E:T ratio of 3:1 for 4 h. The level of TNF-α and IFN-γ in the supernatant was measured by ELISA. A Total of three independent experiments were performed. *P < 0.05, **P < 0.01,***P < 0.001.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Transfection, Reporter Assay, Luciferase, Activity Assay, Biomarker Discovery, RNA Expression, Expressing, Western Blot, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: Cbl-b inhibitors enhance Notch1-positive CD8+ T-cells anti-tumor responses. Organoids were derived from a syngeneic TNBC mouse model, C0321, in FVB mice or immunocompromised athymic Nu/Nu mice and treated with vehicle (control, DMSO) 0.1, 1 and 5µM of NTX-512 or NTX-307, as indicated in the figure. (A) Cancer cell death, (B) production of IFN-gamma in organoid cultures and (C) colocalization of Notch1 and CD8+ T-cells in organoids indicated with white arrows in the panel and in the plot. Panel (C) shows threshold-adjusted images to better highlight Notch1 staining in CD8+ T-cells, whereas the original images are presented in Supplementary Figure 7B . Scale bars length is indicated above each bar (µm). The graphs show averages ± standard deviation from ≥ 10 organoids from at least three independent experiments. *p < 0.05, **p < 0.01, two tailed T-test with equal variance. NS, non-significant.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Derivative Assay, Control, Staining, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: A2AR modulates Notch1 degradation and T-cell functions. Primary CD8+ T-cell isolated from the spleen and lymph nodes of C57B16 or FVB mice were stimulated with anti-CD3/CD28 antibodies and treated with vehicle (control, DMSO) or 1µM ZM-241385 (ZM) or 1µM CGS-21680 (CGS) or ZM+CGS, as indicated in the figure. (A) Protein levels of Notch1 full length (N1FL), Notch1 transmembrane (N1TM) and Notch1 transcriptionally active form (NICD) in primary activated CD8+ T-cells. (B) Ubiquitinated Notch1 protein levels from Notch1 immunoprecipitation in primary activated CD8+ T-cells. Densitometry (O.D.) results for ubiquitinated Notch1 normalized by IP:N1TM are shown below the panel. Negative control refers to samples immunoprecipitated using beads but not antibody. (C) Notch1 protein levels over time in primary activated CD8+ T-cells in the presence of the protein synthesis inhibitor Cycloheximide (CHX). The plot shows densitometry results of western blots. (D) Proliferation and production of IFN-γ in supernatants from samples of primary activated CD8+ T-cells. The graphs show averages ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, two tailed T-test with equal variance. Unstimulated cells (Unstim) and vehicle treated cells (control) were used as controls. β-actin was used to normalize densitometry values.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Isolation, Control, Immunoprecipitation, Negative Control, Western Blot, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: A2AR antagonist ZM enhances Notch1-positive T-cell anti-tumor activity. Organoids were derived from a syngeneic TNBC mouse model, C0321, in FVB mice or immunocompromised athymic Nu/Nu mice and treated with vehicle (control, DMSO) or 1µM ZM-241385 (ZM) or 1µM CGS-21680 (CGS) or ZM+CGS, as indicated in the figure. (A) Colocalization of Notch1 and CD8+ T-cells and production of IFN-gamma in organoids. (B) Growth of organoids measured over time. (C) Cancer cell death in organoids from immunocompetent vs . immunocompromised mice. Scale bars length is indicated above each bar (µm). The graphs show averages ± standard deviation from ≥ 10 organoids from at least three independent experiments. *p < 0.05, **p < 0.01, two tailed T-test with equal variance. NS, non-significant.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Activity Assay, Derivative Assay, Control, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: A2AR promotes Cbl-b-mediated Notch1 degradation via STS-1. Mass spectrometry analysis was carried out in lysates where Notch1 was immunoprecipitated from primary mouse CD8+ T-cells stimulated with anti-CD3/CD28 and treated with vehicle (control, DMSO) or 1µM ZM-241385 (ZM) or 1µM CGS-21680 (CGS) or ZM+CGS, as indicated in the figure. (A) Heat map of mass spectrometry analysis of Notch1 interactome. Uniprot IDs of the detected proteins are indicated on the right side of the heat map. Protein abundance is expressed as NSAF log2 fold-change on a color scale from blue to red where blue indicates the lowest and red the highest value, respectively. (B) Volcano plot of proteins identified in the mass spectrometry in ZM-treated vs . CGS-treated CD8+ T-cells. The X axis indicates fold change (log2) and Y axis the p-value (-log10). The dotted lines indicate the cut-off separating proteins with p<0.05 (blue and red dots) from non-significant ones (black dots). Proteins that are interacting the least with Notch1 are shifted to the left of the plot whereas the ones interacting the most are shifted to the right. The bottom graph shows the interaction between Notch1 and STS-1 (protein abundance) from the mass spectrometry analysis. (C) Protein levels of Notch1, STS-1 and Cbl-b in primary activated CD8+ T-cells isolated from C57BL6 (WT) or STS-1/2 -/- (STS-1/2 KO) mice and treated with vehicle (control, DMSO) or 1µM CGS. Densitometry (O.D.) results for N1TM and Cbl-b normalized by β-actin are shown below the panel. (D) Top 10 ubiquitin ligases interacting with Notch1 ordered by the most to the least interacting based on mass spectrometry results (protein abundance). The graphs show averages ± standard deviation from three independent experiments. **p<0.01, two tailed T-test with equal variance.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Mass Spectrometry, Immunoprecipitation, Control, Quantitative Proteomics, Isolation, Ubiquitin Proteomics, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: Cbl-b ubiquitinates and degrades Notch1. (A, B) Notch1 or Cbl-b co-immunoprecipitation in protein lysates from 293T cells were transfected with various Myc-tagged Notch1 and/or HA-tagged Cbl-b constructs, as indicated. (C) Ubiquitinated Notch1 protein levels detected upon immunoprecipitation of Notch1 in 293T cells co-transfected with a His-tagged NICD construct and various HA-tagged Cbl-b constructs, as indicated. (D) Notch1 activation in 293T cells measured using the Hes-luciferase reporter gene system. (E) Protein levels of Notch1 and IFN-gamma production in primary activated CD8+ T-cells isolated from C57BL6 (WT) or Cbl-b -/- (Cbl-b KO). Densitometry (O.D.) results for N1TM normalized by β-actin are shown below the panel. The graphs show averages ± standard deviation from three independent experiments. **p < 0.01, ***p < 0.001 two tailed T-test with equal variance. Negative control refers to samples immunoprecipitated using rabbit IgG instead of anti-Notch1/HA-tag/Cbl-b antibody. None, non-transfected cells. EV, empty vector.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Immunoprecipitation, Transfection, Construct, Activation Assay, Luciferase, Isolation, Standard Deviation, Two Tailed Test, Negative Control, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: Genetic KO and pharmacologic inhibition of Cbl-b rescue Notch1 and T-cell functions from A2AR-mediated immunosuppression. (A) Protein levels of Notch1 and (B) IFN-gamma production in primary activated CD8+ T-cells isolated from C57BL6 (WT) or Cbl-b -/- (Cbl-b KO). Densitometry (O.D.) results for N1TM normalized by β-actin are shown below the panel. (C) Protein levels of Notch1 in primary activated CD8+ T-cells and treated with Cbl-b inhibitors (1µM NTX-512, NTX-447, and NTX-307) or 1µM ZM-241385 (ZM) or 1µM CGS-21680 (CGS) or combinations, as indicated in the figure. (D) Proliferation, (E) IFN-γ and Granzyme B in primary CD8+ T-cells treated as in (C) . (F) IFN-γ production in unmodified or CRISPR-Cas9 Notch1 KO primary activated CD8+ T-cells untreated (control) or treated with 1µM NTX-307. The panel in (F) shows Notch1 protein levels in unmodified vs . Notch1 KO cells. The graphs show averages ± standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. two tailed T-test with equal variance. N.S, non-significant.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Inhibition, Isolation, CRISPR, Control, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: Cbl-b-Notch1 axis regulation. Adenosine induces immunosuppression in CD8+ T-cells in the tumor microenvironment by activating the adenosine A2A receptor (A2AR), thus causing downregulation of Notch1 and suppression of effector functions. A2AR controls Notch1 levels by regulating the ubiquitin ligase Casitas B-lineage lymphoma b (Cbl-b), which mediates the ubiquitination and degradation of Notch1. A2AR promotes Cbl-b via Suppressor of T-cell receptor signaling 1 (STS-1) Tyr-phosphatase, possibly through Tyr-dephosphorylation of Cbl-b. A2AR, together with other receptors, including T-cell receptor (TCR) and co-stimulation receptor CD28, may control Notch1 levels in T-cells and, in turn, effector functions by modulating Cbl-b. This model places Cbl-b at the core of a regulatory axis, which, by integrating positive and negative signals from different receptors, determines the fate of Notch1 and effector functions. Pharmacological inhibition of Cbl-b blocks this axis and reactivates Notch1 and effector functions.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: Ubiquitin Proteomics, De-Phosphorylation Assay, Control, Inhibition

Journal: Frontiers in Immunology

Article Title: Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses

doi: 10.3389/fimmu.2022.987298

Figure Lengend Snippet: Reactivation of Notch1 in T-cells overcomes immunosuppression. Immunosuppressive signals induce suppression in CD8+ T-cells in the tumor microenvironment. Some of these signals, including adenosine, may suppress CD8+ T-cell effector functions by downregulation of Notch1. Reactivation of Notch1, with Cbl-b inhibitors or other strategies, may tune CD8+ T-cells against immunosuppression and enhance effector functions, ultimately promoting anti-cancer responses in the tumor microenvironment.

Article Snippet: The following primary antibodies were used for Western blot in this study: anti-Notch1 (D1E11, Cell Signaling, or mN1A, Novus, or C-20, SCBT) for Notch1 full length and cleaved forms; anti-cleaved Notch1 Val1744 (D3B8, Cell Signaling or PA5-99448, Invitrogen-Thermofisher) for NICD; anti-Cbl-b (G1, Santa Cruz Biotechnology or 12781-1-AP, Proteintech); anti-STS-1 (19563-1-AP, Proteintech); anti-β-actin (AC-15, SCBT); anti-phospho-Tyr (PY99, STCB); anti-HA tag (Clone 6E2, Millipore).

Techniques: