rabbit anti human cleaved notch1  (Cell Signaling Technology Inc)

Journal: Biomedicines

Article Title: Analysis of Primary Chronic Lymphocytic Leukemia Cells’ Signaling Pathways

doi: 10.3390/biomedicines12030524

Figure Lengend Snippet: qPCR analysis of CLL samples for the mRNA expression of NOTCH1, DELTEX1, HES1, and AIOLOS. cDNA was prepared from RNA isolated from blood samples of CLL patients and compared with normal CD19 + peripheral B cells. Relative expression levels were normalized with GUS gene expression. Data are expressed as 2 −ΔCT . Mean values and SEM of replicates are shown. The values of the control samples are shown as a solid line.

Article Snippet: Briefly, 5 μg of proteins from each sample were separated using SDS-PAGE on a 10% gel, transferred to a nitrocellulose membrane, blocked in Blotto buffer for one hour, and incubated overnight with primary antibodies: Rabbit anti-human AIOLOS, Rabbit anti-human cleaved NOTCH1, Rabbit anti-human NOTCH1, and Rabbit anti-human β-ACTIN (all from Cell Signaling Technology, Inc., Beverly, MA, USA) [ , ].

Techniques: Expressing, Isolation, Gene Expression, Control

Journal: Biomedicines

Article Title: Analysis of Primary Chronic Lymphocytic Leukemia Cells’ Signaling Pathways

doi: 10.3390/biomedicines12030524

Figure Lengend Snippet: Correlation plot of data from gene expression ( top ) and protein analysis ( bottom ) of CLL samples for NOTCH1, HES1, DELTEX, and AIOLOS. Correlation coefficients and p -values are indicated.

Article Snippet: Briefly, 5 μg of proteins from each sample were separated using SDS-PAGE on a 10% gel, transferred to a nitrocellulose membrane, blocked in Blotto buffer for one hour, and incubated overnight with primary antibodies: Rabbit anti-human AIOLOS, Rabbit anti-human cleaved NOTCH1, Rabbit anti-human NOTCH1, and Rabbit anti-human β-ACTIN (all from Cell Signaling Technology, Inc., Beverly, MA, USA) [ , ].

Techniques: Gene Expression

Journal: Biomedicines

Article Title: Analysis of Primary Chronic Lymphocytic Leukemia Cells’ Signaling Pathways

doi: 10.3390/biomedicines12030524

Figure Lengend Snippet: Gene and protein expression profile of primary leukemia samples in peripheral blood and bone marrow. ( A ) Ten B-CLL cells, collected from peripheral blood (PBL) and bone marrow (BM), were analyzed for NOTCH1, DELTEX1, HES1, and AIOLOS gene expression using qRT-PCR. Ct values were normalized to the expression of the GUSB housekeeping gene, and expressed as relative gene expression. ( B ) The protein expression of NOTCH1, HES1, and AIOLOS was analyzed using flow cytometry after the labelling of cells with the corresponding antibodies.

Article Snippet: Briefly, 5 μg of proteins from each sample were separated using SDS-PAGE on a 10% gel, transferred to a nitrocellulose membrane, blocked in Blotto buffer for one hour, and incubated overnight with primary antibodies: Rabbit anti-human AIOLOS, Rabbit anti-human cleaved NOTCH1, Rabbit anti-human NOTCH1, and Rabbit anti-human β-ACTIN (all from Cell Signaling Technology, Inc., Beverly, MA, USA) [ , ].

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Flow Cytometry

Journal: bioRxiv

Article Title: Mutational order and epistasis determine the consequences of FBXW7 mutations during colorectal cancer evolution

doi: 10.1101/2023.08.25.554836

Figure Lengend Snippet: Characterisation of W and F organoids. a. Brightfield microscopy of W and F organoids revealed no observable phenotypic differences. Scale bars represent 500μm. b. Western blot validation showing loss of FBXW7 protein in F organoids. c. Immunofluorescence of W and F organoids with DAPI nuclear stain (blue), F-actin (red), FBXW7 (orange). Scale bars represent 100μm. d. FBXW7 targets the phosphorylated substrates and ubiquitinates these substrates for proteasomal degradation. This included phosphorylated cJun, phosphorylated CCNE1, phosphosylated cMyc, and notch intracellular domain (NICD). The non-phosphorylated substrates were affected to varying degrees. While upregulation of cJun, was observed, there was no change in the quantities of CCNE1, cMYC and NOTCH. e. Volcano plot from bulk RNAseq of F vs W organoids revealed minimal differential expression of genes. Only HLA-DQB1 was found to be significantly downregulated, and GSTM1 significantly upregulated in F organoids. All experiments were performed with N = 3 biological replicates.

Article Snippet: Rabbit anti-human FBXW7 antibody (1:2500, BS-8394R, Bioss, USA), rabbit anti-human phosphor-CJUN antibody (1:2500, PA5-40193, Invitrogen, USA), rabbit anti-human CJUN antibody (1:2500, ab40766, Abcam, USA), rabbit anti-human phosphor-CCNE1 antibody (1:2500, ab52195, Abcam, USA), rabbit anti-human CCNE1 antibody (1:2500, ab33911, Abcam, USA), rabbit anti-human phosphor-CMYC (1:2500, ab185655 and ab185656, Abcam, USA), rabbit anti-human CMYC (1:2500, ab32072, Abcam, USA), rabbit anti-human NICD (1:2500, #4147, Cell signalling technologies, USA), rabbit anti-human NOTCH1 (1:2500, ab52627, Abcam, USA), and rabbit anti-human GAPDH (1:2500, ab52627, Abcam, USA) was used.

Techniques: Microscopy, Western Blot, Biomarker Discovery, Immunofluorescence, Staining, Quantitative Proteomics

Journal: Frontiers in Medicine

Article Title: Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease

doi: 10.3389/fmed.2022.897188

Figure Lengend Snippet: Immunofluorescence imaging-based analyses of glomerular sections shows erlotinib administration protects WT and Pod-miR-146a –/– mice from STZ injury via reduction in ErbB4 and EGFR. (A) Representative confocal microscopy images of immunofluorescently labeled glomeruli from WT (top three panels) and Pod-miR146a (bottom three panels) mice treated with vehicle alone (Control), with STZ and vehicle (STZ) or with STZ and erlotinib (STZ Erl). Tissue sections were imaged after staining with DAPI (nuclear marker) and antibodies against ErbB4, EGFR, Notch-1 and Synaptopodin (Synpo, podocyte marker) (as indicated). Scale bar, 50 μm. (B) Bar graphs showing quantification of relative glomerular signal intensity of ErbB4, EGFR and Notch-1 in tissue samples from A. Statistics were performed using two-way ANOVA. Data shown are mean ± SEM ( n = 5/group). * p < 0.05; *** p < 0.001; ns, no significant difference.

Article Snippet: For EGFR, Notch-1 and ErbB4 staining, sections were then incubated with primary antibodies—Rabbit anti-mouse EGFR (Millipore, #06847), mouse anti-mouse ErbB4 (Santa Cruz, #sc-8050) and Rabbit anti-mouse Notch-1 (Rockland, #100-401-407) in the blocking buffer at 4 ° C, overnight.

Techniques: Immunofluorescence, Imaging, Confocal Microscopy, Labeling, Staining, Marker

Journal: Frontiers in Medicine

Article Title: Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease

doi: 10.3389/fmed.2022.897188

Figure Lengend Snippet: Mechanistic model. A diagram showing a mechanistic working model. Podocyte expressed miR-146a represses expression of ErbB4 and Notch-1 during homeostatic conditions, thereby controlling the ErbB4/EGFR and TGFβ 1 signaling pathways. Various external stressors or deletion of miR-146a result in de-repression of ErbB4 and Notch-1, thereby driving the harmful ErbB4/EGFR signaling and inducing TGFβ 1. An autocrine feed-forward loop via TGFβ 1 induces the downstream TGFR/Smad3 signaling, that result in podocyte damage, glomerular injury and proteinuria.

Article Snippet: For EGFR, Notch-1 and ErbB4 staining, sections were then incubated with primary antibodies—Rabbit anti-mouse EGFR (Millipore, #06847), mouse anti-mouse ErbB4 (Santa Cruz, #sc-8050) and Rabbit anti-mouse Notch-1 (Rockland, #100-401-407) in the blocking buffer at 4 ° C, overnight.

Techniques: Expressing

Journal: Cell Reports

Article Title: The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition

doi: 10.1016/j.celrep.2021.110103

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human/rat/mouse NICD - Cleaved Notch1 (Val1744), clone D3B8 , Cell Signaling Technology , Cat# 4147; RRID: AB_2153348.

Techniques: Blocking Assay, Virus, Recombinant, Modification, Reverse Transcription, TaqMan Assay, DNA Library Preparation, Gene Expression, Control, RNA Sequencing, Mutagenesis, Knock-Out, Software, Membrane

Journal: Nature Communications

Article Title: Heterogeneity in VEGFR3 levels drives lymphatic vessel hyperplasia through cell-autonomous and non-cell-autonomous mechanisms

doi: 10.1038/s41467-018-03692-0

Figure Lengend Snippet: DLL4 downregulation and decrease in Notch signaling upon loss of VEGFR3. a qRT-PCR analysis of Notch pathway genes in control (siCTRL) and VEGFR3 siRNA (siVEGFR3)-treated human LECs. Bars represent mean relative expression ( n = 3 biological replicates with n = 2 technical replicates each) ± s.d. b qRT-PCR analysis of Dll4 and Notch1 expression in FACS-sorted LECs from P13-P14 Cre − ( Ctrl ) and Vegfr3 flox/flox ; R26-mTmG ; Prox1-CreER T2 ( R3 fl/fl ) mice. Tamoxifen (150 µg) was administered at P2, P4, and P6. Bars represent mean relative expression ( n = 5 Ctrl and n = 4 R3 fl/fl mice) ± s.d. c Whole-mount immunofluorescence of ear skin of 3 weeks old Vegfr3 flox/flox ; R26-mTmG ; Prox1-CreER T2 and Cre-negative littermate mice treated with Tamoxifen at P2, P4, and P6. Note downregulation of DLL4 protein in targeted GFP + LECs (arrows) compared to non-targeted GFP − LECs (arrowhead) in the mutant ( flox/flox ) ear. d Hey1 , ( e ) Efnb2 and ( f ) Ccnb1 expression analyzed by qRT-PCR in FACS-sorted LECs from P13-P14 Cre − ( Ctrl ) and Vegfr3 flox/flox ; R26-mTmG;Prox1-CreER T2 ( R3 fl/fl ) mice. Results from non-targeted (Tomato + GFP − ; red), and targeted (Tomato − GFP + ; green) LECs are displayed separately. Horizontal lines represent mean relative expression ( n = 5 Ctrl and n = 4 R3 fl/fl mice with n = 2 technical replicates for each). * P < 0.05, ** P < 0.01, *** P < 0.001. Two-tailed unpaired Student’s t test ( a , b , d – f ). Scale bars: 50 µm ( c ). ns: not significant

Article Snippet: Proteins were detected using mouse anti-human VEGFR3 (clone 9D9, MAB3757, Millipore, 1:50), rabbit anti-human Cleaved NOTCH1 (Val1744) (#4147, CST, 1:1000), rabbit anti-human DLL4 (#2589, CST, 1:1000), rabbit anti-human GAPDH (#2118, CST, 1:3000) and mouse anti-human α-tubulin (T5168, Sigma, 1:1000) and visualized by using an ECL chemiluminescent substrate reagent (Invitrogen).

Techniques: Quantitative RT-PCR, Control, Expressing, Immunofluorescence, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Heterogeneity in VEGFR3 levels drives lymphatic vessel hyperplasia through cell-autonomous and non-cell-autonomous mechanisms

doi: 10.1038/s41467-018-03692-0

Figure Lengend Snippet: Global and mosaic loss of DLL4 promote LEC proliferation through inhibition of Notch signaling in neighboring cells. a , b DAPT induced effect on the proliferation of control (siCTRL) or VEGFR3 (siVEGFR3) siRNA-treated LECs. a Tile scan immunofluorescence images and ( b ) quantification of proliferating EdU + LECs after 3 h of incorporation (mean ( n = 3 biological replicates) ± s.e.m). c Western blot analysis of cell lysates from LECs treated with siCTRL or NOTCH1 siRNA (siNOTCH1) and ( d ) quantification of proliferating EdU + LECs cultured with or without VEGF-C (data are normalized to the proliferation rate in siCTRL alone and represent mean ( n = 3 biological replicates) ± s.e.m). e Western blot analysis of cell lysates from LECs treated with siCTRL or DLL4 siRNA (siDLL4) and ( f ) quantification of proliferating EdU + cells in co-cultures of siCTRL and siDLL4 LECs mixed in different ratios (data are normalized to the proliferation rate in siCTRL alone and represent mean ( n = 6 biological replicates) ± s.e.m). g Model of cell-autonomous and non-cell-autonomous effects of VEGFR3 expression defining LEC responses during sprouting lymphangiogenesis. VEGF-C-VEGFR3 signaling positively regulates DLL4 expression leading to activation of Notch and cell cycle arrest in neighboring LECs. VEGFR3 deficiency (green cell) consequently leads to downregulation of DLL4 and inhibition of Notch in neighboring LECs, but only VEGFR3 expressing (red) cells respond by proliferation and sprouting. When VEGFR3 − cells are in excess, VEGFR3 + cells interact predominantly with LECs with low DLL4 and respond by proliferation (dashed line) * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Two-tailed unpaired Student’s t test ( b , d , f ) or one-way ANOVA with Tukey’s post hoc test ( d ; comparison between VEGF-C untreated and treated groups). Scale bars: 200 µm ( a ). ns: not significant

Article Snippet: Proteins were detected using mouse anti-human VEGFR3 (clone 9D9, MAB3757, Millipore, 1:50), rabbit anti-human Cleaved NOTCH1 (Val1744) (#4147, CST, 1:1000), rabbit anti-human DLL4 (#2589, CST, 1:1000), rabbit anti-human GAPDH (#2118, CST, 1:3000) and mouse anti-human α-tubulin (T5168, Sigma, 1:1000) and visualized by using an ECL chemiluminescent substrate reagent (Invitrogen).

Techniques: Inhibition, Control, Immunofluorescence, Western Blot, Cell Culture, Expressing, Activation Assay, Two Tailed Test, Comparison