anti notch1 antibody  (R&D Systems)

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/eLife.98747

Figure Lengend Snippet: ( a–c ) Representative triple stain images for N1ICD, Notch1, and Jag2 mRNA in sebaceous glands (SGs) from mice (n=5 each) treated with aRW, 3 d post-treatment. ( a ) Four channels showing N1ICD, N1 ISH, J2 ISH, and DAPI. ( b ) Two channels showing N1 ISH and J2 ISH. ( c ) One channel showing N1ICD. ( d-f ) Representative triple stain images for N1ICD, Notch1, and Jag2 mRNA in SGs from mice (n=5 each) treated with aJ1, 3 d post-treatment. ( d ) Four channels showing N1ICD, N1 ISH, J2 ISH, and DAPI. ( e ) Two channels showing N1 ISH and J2 ISH. ( f ) One channel showing N1ICD. ( g ) Quantification of the percentage of N1ICD positive (N1ICD+) basal stem cells in SGs from mice (n=5 each) treated with aRW, aJ1, and aJ2, 3 d post-treatment. Percentage was calculated by dividing the number of N1ICD+ basal stem cells by the total number of basal stem cells in each SG. ( h–j ) Representative triple stain images for N1ICD, Notch1, and Jag2 mRNA in SGs from mice (n=5 each) treated with aJ2, 3 d post-treatment. ( h ) Four channels showing N1ICD, N1 ISH, J2 ISH, and DAPI. ( i ) Two channels showing N1 ISH and J2 ISH. ( j ) One channel showing N1ICD. ( k ) Quantification of what percentage of the N1ICD+ basal stem cells express both N1 ISH and J2 ISH, only N1 ISH, or only J2 ISH. ( g and k ) Total n of SGs quantified per treatment: aRW=13, aJ1=15, aJ2=15. Error bars represent SEM. Scale bars are 25 μm. Figure 2—source data 1. Source data for .

Article Snippet: Antibody , Anti-Notch1 (Human monoclonal) , Genentech , , Inhibiting antibody (5 mg/kg).

Techniques: Staining

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/eLife.98747

Figure Lengend Snippet: (Related to ). ( a–c ) Quantification of the location of N1ICD+ basal stem cells in sebaceous glands (SGs) from mice (n=5 each) treated with aRW ( a ), aJ1 ( b ), and aJ2 ( c ), 3 d post-treatment. The SG was divided into five sections along the proximal-distal axis. The number of N1ICD+ basal stem cells in each section was divided by the total number of N1ICD+ basal stem cells in each SG. Total n of SGs quantified per treatment: aRW=13, aJ1=15, aJ2=15. ( d ) Quantification of what percentage of the total basal stem cells (N1ICD+ and N1ICD-) express both N1 ISH and J2 ISH. Total n of SGs quantified per treatment: aRW=13, aJ1=15, aJ2=15. ( e,f ) Representative triple stain images for N1ICD, Notch1, and Jag2 mRNA in SGs from mice (n=5 each) treated with ( e ) aRW and ( f ) aJ2, 3 d post-treatment. ( g ) Quantification of the width of the interfollicular epidermis (IFE), 7 d after treatment with aRW, aJ1, aJ2, and aJ1J2. p-values: aJ1=0.528, aJ2=0.885, aJ1J2=0.840. Total n of SGs quantified per treatment: aRW=26, aJ1=27, aJ2=27, aJ1J2=22. ( h ) Quantification of the width of the adipocyte layer, 7 d after treatment with aRW, aJ1, aJ2, and aJ1J2. p-values: aJ1=0.890, aJ2=0.948, aJ1J2=0.374. Total n of SGs quantified per treatment: aRW=13, aJ1=13, aJ2=15, aJ1J2=15. ( i,j ) Representative hematoxylin and eosin (H&E) images of SGs from mice (n=5 each) treated with aRW ( i ) and aJ2 ( j ), 7 d post-treatment. Student’s t-test used for statistical analysis. Error bars represent SEM. Scale bars are 100 μm. Figure 2—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Anti-Notch1 (Human monoclonal) , Genentech , , Inhibiting antibody (5 mg/kg).

Techniques: Staining

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/eLife.98747

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Notch1 (Human monoclonal) , Genentech , , Inhibiting antibody (5 mg/kg).

Techniques: Control, Staining, Sequencing, RNAscope, Multiplex Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO sustains CSC stemness through the activation of Notch1. (A) Immunoblot analysis of Notch1 and Hes1 protein in non‐CSCs and CSCs. (B) The protein expression of Notch1 was analysed in non‐CSCs treated with DETA NONOate (20 μM) for 24 h. (C) CSCs were treated with L‐NAME for 24 h and detected for Notch1 protein with immunoblots. (D, E) CSCs were treated with L‐NAME (100 μM)/1400 W (100 μM) in the presence or absence of the DETA NONOate (20 μM)/GSNO (200 μM) for 24 h. Protein expression of Notch1 and Hes1 were detected by immunoblot. (F–H) CSCs were exposed to L‐NAME (100 μM) with or without the reintroduction of NICD and were analysed for expression of Notch1 protein, stem‐related transcripts and spheres formation. Mean ± SEM from 5 to 9 independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 with paired t ‐test (B, C), unpaired t ‐test (A) and ANOVA plus Turkey's method (D, G, H).

Article Snippet: Cells were treated with BD Fix Buffer I and Cytoperm Buffer and were stained with the following antibodies: PE‐conjugated anti‐CD133 antibody (Invitrogen, 12‐1338‐42), anti‐human Notch1 antibody (Santa Cruz Biotechnology, sc‐373891) plus goat anti‐mouse IgG (DyLight 649, Abbkine, A23610).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO inhibits the ubiquitination of Notch1. (A) mRNA level of Notch1 was measured in CSCs with or without L‐NAME (100 μM) treatment for 24 h. Mean ± SEM from four independent experiments. (B) Notch1 cleavage‐related mRNA expressions in non‐CSCs and CSCs was determined with qPCR. Mean ± SEM from six independent experiments. (C) CSCs were treated with or without L‐NAME (100 μM) for 24 h and detected for Notch1 cleavage‐related mRNA expressions by qPCR. Mean ± SEM from 6 to 8 independent experiments. (D) CSCs were treated with L‐NAME (100 μM) in the presence or absence of the MG132 (10 μM) for 4 h. Protein expression of Notch1 was detected by immunoblot. Mean ± SEM from four independent experiments. (E) Notch1 ubiquitination in non‐CSCs and CSCs was analysed using endogenous immunoprecipitated Notch1 protein. * p < 0.05, ** p < 0.01 with paired t ‐test (A, C), unpaired t ‐test (B) and ANOVA plus Turkey's method (D).

Article Snippet: Cells were treated with BD Fix Buffer I and Cytoperm Buffer and were stained with the following antibodies: PE‐conjugated anti‐CD133 antibody (Invitrogen, 12‐1338‐42), anti‐human Notch1 antibody (Santa Cruz Biotechnology, sc‐373891) plus goat anti‐mouse IgG (DyLight 649, Abbkine, A23610).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO inhibits the ubiquitination of Notch1 by facilitating its interaction with UCHL1. (A) Potential DUBs and E3 ligase of Notch1 obtained from the UbiBrowser database. (B, C) DUBs and E3 ligase mRNA expressions in non‐CSCs and CSCs were determined with qPCR. Mean ± SEM from six independent experiments. (D) Immunoblot analysis of UCHL1 protein in non‐CSCs and CSCs. Mean ± SEM from five independent experiments. (E) Co‐immunoprecipitation analysis of Notch1‐UCHL1 interaction in non‐CSCs and CSCs. (F) Genetic knockdown efficiency of UCHL1 in CSCs by lentiviral shRNA transfections. Mean ± SEM from four independent experiments. (G) Immunoblot analysis of Notch1 protein in CSCs transfected with or without the independent UCHL1 shRNAs. Mean ± SEM from four independent experiments. (H) Genetic knockdown efficiency of WWP1, MDM2, USP9X and PSMD7 in CSCs by lentiviral shRNA transfections. Mean ± SEM from 4 to 6 independent experiments. (I) Immunoblot analysis of Notch1 protein in CSCs transfected with or without the independent WWP1, MDM2, USP9X and PSMD7 shRNAs. (J) Immunoprecipitation analysis of ubiquitination of Notch1 in CSCs that were transfected with shUCHL1. (K) CSCs were transfected with UCHL1 shRNAs in the presence or absence of the DETA NONOate (20 μM). The protein of Notch1 was analysed with immunoblot. Mean ± SEM from four independent experiments. (L) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs that were transfected with UCHL1 shRNAs together with DETA NONOate (20 μM). (M) CSCs transfected with UCHL1 shRNAs were analysed for expression of stem‐related transcripts. Mean ± SEM from 5 to 6 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 with paired t ‐test (M) and ANOVA plus Turkey's method (F–H, K).

Article Snippet: Cells were treated with BD Fix Buffer I and Cytoperm Buffer and were stained with the following antibodies: PE‐conjugated anti‐CD133 antibody (Invitrogen, 12‐1338‐42), anti‐human Notch1 antibody (Santa Cruz Biotechnology, sc‐373891) plus goat anti‐mouse IgG (DyLight 649, Abbkine, A23610).

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Knockdown, shRNA, Transfection, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO‐facilitated s‐nitrosylation of Notch1 promotes its binding to UCHL1. (A) Biotin switch assay was employed to detect the SNO‐Notch1 levels in both non‐CSCs and CSCs. (B) The SNO‐Notch1 level in DETA NONOate‐treated (20 μM, 24 h) CSCs were detected by biotin switch assay. (C) The SNO‐Notch1 level in L‐NAME‐treated (100 μM, 24 h) CSCs were detected by biotin switch assay. (D) Notch1 protein levels in CSCs with or without ODQ (10 μM, 24 h) were analysed by immunoblotting. Mean ± SEM from four independent experiments. (E) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs in the presence or absence of L‐NAME (100 μM) for 24 h. (F) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs treated with DETA NONOate (20 μM) for 24 h. (G) Co‐immunoprecipitation analysis of Notch1 and UCHL1 interaction in CSCs in the presence or absence of DETA NONOate (20 μM). (H) Co‐immunoprecipitation analysis of Notch1 and UCHL1 interaction in CSCs with or without L‐NAME (100 μM).

Article Snippet: Cells were treated with BD Fix Buffer I and Cytoperm Buffer and were stained with the following antibodies: PE‐conjugated anti‐CD133 antibody (Invitrogen, 12‐1338‐42), anti‐human Notch1 antibody (Santa Cruz Biotechnology, sc‐373891) plus goat anti‐mouse IgG (DyLight 649, Abbkine, A23610).

Techniques: Binding Assay, Biotin Switch Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: Targeting UCHL1 and NO downregulates the expression levels of Notch1 and CD133. (A) Representative of PDO‐primary tumour pair with immunostaining for CD31. Nuclei were stained with Hoechst. Scale bar, 20 μm. (B) Representative of PDO‐primary tumour pair with immunostaining for PanCK. Nuclei were stained with Hoechst. Scale bar, 20 μm. (C) Representative HE staining of PDO‐primary tumour pairs. Scale bars, 100 μm. (D, E) PDOs treated with or without DETA NONOate (20 μM) were analysed for CD133 and Notch1 protein. Representatives from six independent experiments. (F, G) PDOs were transfected with UCHL1 shRNAs in the presence or absence of the DETA NONOate (20 μM), followed by analysis of CD133 and Notch1 protein. Representatives from 4 to 5 independent experiments. (H, I) PDOs exposed to 10 Gy x‐rays were treated with L‐NAME (100 μM), followed by analysis of CD133 and Notch1 protein. Representatives from 3 to 5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with paired t ‐test (D, E) and ANOVA plus Turkey's method (F–I).

Article Snippet: Cells were treated with BD Fix Buffer I and Cytoperm Buffer and were stained with the following antibodies: PE‐conjugated anti‐CD133 antibody (Invitrogen, 12‐1338‐42), anti‐human Notch1 antibody (Santa Cruz Biotechnology, sc‐373891) plus goat anti‐mouse IgG (DyLight 649, Abbkine, A23610).

Techniques: Expressing, Immunostaining, Staining, Transfection

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: 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: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 3. Requirement of the ligand-binding domain of Notch1 for the activation and increased cell surface expression of Notch1 by transient co-transfection of TM2D3. (a, b) Activation of Notch1 with a full-length ectodomain but not those that lack EGF repeats (Notch1 LNR, LNR CC > SS, and ΔE) (Supplementary Fig. S5 online) by co-transfection of TM2D3. Cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoblotting was performed with the indicated antibodies. Notch1 LNR CC > SS and ΔE are constitutively active. (c) Inability of TM2D3 to activate Notch1 that lacks EGF repeats 11 and 12. Experiments were conducted as in a and b. (d, e) Increased Notch1 expression at cell surface by co-transfection of TM2D3 and its independence on EGF repeats 11 and 12. Cells transfected with vectors for the indicated proteins or empty vector (Vector) as a control were incubated with or without a non-membrane permeable biotinylation reagent as indicated. After fractionation with an avidin agarose, immunoblotting was performed with the indicated antibodies. (f) Cell surface expression of TM2D3. Experiments were conducted as in (d) and (e).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Ligand Binding Assay, Activation Assay, Expressing, Cotransfection, Transfection, Plasmid Preparation, Control, Western Blot, Incubation, Membrane, Fractionation, Avidin-Biotin Assay

Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 4. Physical association of Notch1 and TM2D3. (a) Physical association of TM2D3 and Notch1. 293 T cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoprecipitation (IP) and immunoblotting were performed with the indicated antibodies. (b, c) Requirement of NRR of Notch1 for physical association with TM2D3. Experiments were conducted as in (a). (d) Requirement of LNR-A of NOTCH1 for physical association with TM2D3. A schematic diagram of NOTCH1 constructs used is shown in Supplementary Fig. S5 online. Experiments were conducted as in a. (e) Requirement of C-terminal portion including transmembrane 2 domain of TM2D3 for the activation of Notch1. Experiments were conducted as in (a). (f) No dependency on any one region of TM2D3 for the physical association with Notch1. Experiments were conducted as in (b).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Construct, Activation Assay

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