Journal: International Immunology

Article Title: Kit-independent mast cell adhesion mediated by Notch

doi: 10.1093/intimm/dxy067

Figure Lengend Snippet: Adhesion of MCs to Notch ligand-transduced OP9 stromal cells was not inhibited by the insufficiency of Kit in MCs. (A) WT MCs were cultured on each OP9 stromal cell with 5 µg ml−1 of anti-IL-7Rα mAb (Ctrl mAb) or anti-Kit mAb (ACK2). The percentages of adherent MCs at each incubation time (5, 15, 30, 60 and 120 min) are shown. Data are the mean ± SE of triplicate cultures (*P < 0.05 between Ctrl mAb and anti-Kit mAb at each time point, two-tailed Welch’s t-test). (B) Surface expression of Kit and FcɛRIα on MCs-Kit +/+ and -Kit W-sh/W-sh . (C) Percentages of adherent MCs-Kit +/+ and -Kit W-sh/W-sh on each OP9 stromal cell at 60 min of the adhesion assay are shown (mean ± SE of triplicate cultures; *P < 0.05, two-tailed Welch’s t-test). (D) Surface expression of Notch1 and Notch2 on MCs-Kit +/+ and -Kit W-sh/W-sh , and Kit+ peritoneal MCs (see Supplementary Figure 2). (E) The expression of Kit and Fcer1a in the mixed lysates of adherent MCs-Kit +/+ or -Kit W-sh/W-sh and each OP9 stromal cell after 60 min of the adhesion assay was analyzed by RT–PCR. RT–PCR with total RNAs from each MC alone before the adhesion assay was designated as ‘Pre’. (F) The expression of Kit, Fcer1a and Kitl in each OP9 stromal cell was analyzed by RT–PCR. (E, F) Original gel sources and negative controls are in Supplementary Figure 1.

Article Snippet: Regarding the detection of Notch receptors in , cells were stained with fluorescein-labeled mAbs and sheep control, anti-mouse Notch1 or anti-mouse Notch2 pAbs (R&D Systems), and then with rabbit anti-sheep IgG-biotin (Vector Laboratories) and streptavidin-PE (SouthernBiotech).

Techniques: Cell Culture, Incubation, Two Tailed Test, Expressing, Cell Adhesion Assay, Reverse Transcription Polymerase Chain Reaction

Journal: International Immunology

Article Title: Kit-independent mast cell adhesion mediated by Notch

doi: 10.1093/intimm/dxy067

Figure Lengend Snippet: Spreading of adherent MCs-Kit W-sh/W-sh on Notch ligand-transduced OP9 stromal cells was dependent on the Notch receptor–ligand interaction, but independent of γ-secretase-mediated Notch signaling. (A) Adhesion assays (60 min) for MCs-Kit W-sh/W-sh on each OP9 cell with control pAb (20 µg ml−1) or anti-Notch1 pAb plus anti-Notch2 pAb (10 µg ml−1 each). Cultures contained 76.9 µM NaN3, which had no effect on the adhesion of MCs. (B) MCs-Kit W-sh/W-sh were pre-treated with the γ-secretase inhibitor DAPT (50 µM) or its solvent DMSO (0.5% vol/vol) for 30 min, and the adhesion assay (60 min) was performed with the reagents. (C) MCs-Kit +/+ were pre-treated with an anti-Kit mAb (ACK2, 10 µg ml−1) plus DAPT (30 µM) or DMSO (0.3% vol/vol) for 10 min, and the adhesion assay (60 min) was performed with the reagents. Data are the mean ± SE of triplicate cultures. Asterisks denote the significantly different responses of each adhesion state from control treatments (*P < 0.05, two-tailed Welch’s t-test).

Article Snippet: Regarding the detection of Notch receptors in , cells were stained with fluorescein-labeled mAbs and sheep control, anti-mouse Notch1 or anti-mouse Notch2 pAbs (R&D Systems), and then with rabbit anti-sheep IgG-biotin (Vector Laboratories) and streptavidin-PE (SouthernBiotech).

Techniques: Control, Solvent, Cell Adhesion Assay, Two Tailed Test

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Schematic representation of reporter and mNotch-1-IC-specific expression constructs. (A) The luciferase construct pGa981/6 carries 12 RBP-Jκ binding sites upstream of a minimal β-globin promoter. Abbreviations: H3, HindIII, N, NsiI, X, XhoI. (B) The mammalian Notch-1 receptor and the derived deletion proteins used for expression. All constructs carry an N-terminal FLAG tag (open circle). The first and last amino acids compared to the full-length protein are indicated in parentheses. The −ΔEP mutants represent in-frame deletions of aa 2098 to 2112. The asterisk designates a 1758 WFP/LAA 1760 mutation in the mRAM23 domain. Abbreviations: SS, signal sequence, EGF, epidermal growth factor-like repeats, LNR, Lin/Notch repeats, TM, transmembrane domain, RAM, RAM23 domain, ANK, ankyrin repeats, NLS, nuclear localization signal.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Expressing, Construct, Luciferase, Binding Assay, Derivative Assay, FLAG-tag, Mutagenesis, Sequencing

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Notch-1-IC-mediated transcription depends on the primary RBP-Jκ binding site within the RAM domain. Portions (2 μg) of reporter construct pGa981/6 were cotransfected into HeLa cells together with the indicated amounts of expression plasmids. Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and standard deviations from at least four independent experiments are shown.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Binding Assay, Construct, Expressing, Luciferase, Activity Assay

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Dominant negative effect of mNotch-1-ICΔRBP. Portions (2 μg) of reporter construct pGa981/6 were cotransfected into HeLa cells together with increasing amounts of a Notch-1-IC expression plasmid and the indicated amount of pcDNA3 (open circles) or pcDNA3mN1-ICΔRBP (solid circles). Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and standard deviations from three independent experiments are shown.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Dominant Negative Mutation, Construct, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Detection of RBP/Notch-1 DNA binding complexes. In cell extracts from HEK-293 cells, an RBP-Jκ-specific DNA binding complex can be detected (complex A, lanes 1 and 2). In cell extracts from HEK-293 cells transfected with expression plasmids for the indicated Notch-1 proteins, the RBP-Jκ-specific DNA binding activity was decreased and more slowly migrating complexes appeared (lanes 3 to 12). Transcriptionally active Notch-1 proteins form a slowly migrating complex (complex C, lanes 3, 5, and 7). Treatment with an anti-FLAG antibody (α-FLAG) recognized complex C and supershifted to a novel complex D (lanes 4, 6, and 8). Transcriptionally inactive Notch-1 proteins form a faster-migrating complex (complex E, lanes 9 and 11). In cell extracts from HEK-293 cells transfected with the Notch-1 mutant mNotch-1-P/XB, both complexes (C and D, lanes 7) were formed. Complex E interfered with the anti-FLAG antibody (lanes 8, 10, and 12). Cell-free synthesized RBP-2N was used as a control for RBP DNA binding. The RBP-specific DNA binding activity (A) (lane 13) was recognized by an RBP-specific antibody (α-RBP) to form a supershifted complex B (lane 14). The asterisk designates a nonspecific complex (lanes 3 to 12). The 32P-labeled oligonucleotide SL233 was used as a probe.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Binding Assay, Transfection, Expressing, Activity Assay, Mutagenesis, Synthesized, Labeling

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: (A to C) Transcriptional activity of Notch-1–GFP fusion proteins. (A) Schematic representation of Notch-1 deletion mutants fused to GFP. For details, see Fig. ​Fig.1B.1B. (B) Cell extracts were prepared 24 h after transfection of 5-μg portions of plasmid expressing Notch-1 proteins and Notch-1-specific GFP fusion proteins. Expression was assayed by Western blotting using an anti-FLAG antibody. (C) Stimulation of the reporter construct pGa981/6 by Notch-1–GFP fusion proteins depends on the EP domain. Portions (2 μg) of reporter construct pGa981/6 were cotransfected into HeLa cells with 150, 200, or 250 ng of plasmid expressing the indicated mNotch-1-GFP fusion proteins. Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and standard deviations of four independent experiments are shown. (D) Subcellular localization of Notch-1-GFP fusion proteins in vivo. HEK-293 cells were transfected with plasmids expressing GFP (CMV-GFP) (a) or the Notch-1-IC-derived GFP fusion proteins mNotch-1-IC-GFP (b), mNotch-1-ΔRBP-GFP (c), mNotch-1-P/XB-GFP (d), mNotch-1-E/XB-GFP (e), or mNotch-1-Eh/XB-GFP (f). At 24 h after transfection, the living cells were assayed for GFP expression by fluorescence microscopy. Magnification, ×630.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Construct, Luciferase, In Vivo, Derivative Assay, Fluorescence, Microscopy

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Repression of Notch-1-mediated transactivation by E1A12S and p53. (A and B) Portions (2 μg) of reporter construct pGa981/6 were cotransfected into HeLa cells with 100 ng of plasmid expressing mNotch-1-IC+OP (A) or mNotch-1-IC (B), together with increasing amounts (0.2, 0.4, and 0.6 μg) of plasmids expressing the indicated E1A proteins. (C) Portions (2 μg) of reporter construct pGa981/6 were cotransfected with 100-ng portions of plasmid expressing mNotch-1-IC+OP together with increasing amounts (0.2, 0.4, and 0.6 μg) of plasmids expressing the indicated human and murine p53 proteins. Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and standard deviations from four independent experiments are shown.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Construct, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Notch-1-IC interacts with the CH3 region of p300. (A) Schematic representation of p300-derived protein fragments fused to GST. After bacterial expression, the GST fusion proteins were used in pull-down experiments. The first and last amino acids compared to the full-length protein are indicated in parentheses. (B) Cell-free synthesized E1A protein interacts with various p300 fragments. (C) Cell-free synthesized Notch-1-IC protein interacts with p300 fragment B. (D) Cell-free synthesized p300 interacts with GSTmNotch-1-IC. C-terminally truncated p300 (aa 1 to 1302) fails to interact with GST-Notch-1-IC. GST proteins were immobilized with Sepharose beads and incubated with in vitro-translated and radiolabeled proteins. After extensive washing steps, the reaction mixtures were boiled and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The positions of molecular size markers are shown.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Derivative Assay, Expressing, Synthesized, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: (A) Notch-1-IC/p300 interaction correlates with transcriptional activity. (A) GST p300 fragment B was immobilized with Sepharose beads and incubated with in vitro-translated Notch-1-IC deletion mutants (TNT-N1-) or E1A protein as a control. After extensive washing steps, the reaction mixtures were boiled and proteins were separated by SDS-polyacrylamide gel electrophoresis. Relative transcriptional activity of the Notch-1 proteins in cotransfection experiments is shown (Fig. ​(Fig.4).4). ++, strong transcriptional activation; +, reduced activation; −, loss of activation. (B) Relative binding of in vitro-translated Notch-1-IC deletion mutants to GST-p300 fragment B obtained from densitometric analysis. Exposed films from six experiments were scanned and analyzed by the NIH Image software.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Activity Assay, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis, Cotransfection, Activation Assay, Binding Assay, Software

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Notch-1/p300 interaction in vivo. Expression of endogenous p300, mNotch-1-IC+OP (lane 1), and mNotch-1-IC+OP-ΔEP (lane 2) in transiently transfected HEK-293 cells is shown. The amount of p300 protein in cell lysates (lane 3) decreased after incubation with the anti-p300 antibody (lane 4) and accumulated in the precipitate (lane 6). Increasing amounts of blocking peptide added to the reaction mixture (lanes 8 and 9) prevented immunoprecipitation of p300 as well as coimmunoprecipitation of mNotch-1-IC+OP (lane 7). Deletion of the EP domain destabilizes Notch-1/p300 interaction (lanes 10 to 15). Increasing amounts of LiCl in the washing buffer did not interfere with p300 binding to mNotch-1-IC+OP but destabilized p300 binding to mNotch-1-IC+OP-ΔEP (compare lanes 10 to 12 with lanes 13 to 15). Extracts were incubated with agarose beads alone (lane 5) or an agarose-conjugated anti-p300 antibody (lanes 6 to 15). The mixture was divided into three aliquots, and the beads were washed three times with CHAPS lysis buffer containing 150 mM (lanes 10 and 13), 500 mM (lanes 11 and 14), or 1000 mM LiCl (lanes 12 and 15). After a further washing step with CHAPS lysis buffer, the proteins were analyzed by Western blotting.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: In Vivo, Expressing, Transfection, Incubation, Blocking Assay, Immunoprecipitation, Binding Assay, Lysis, Western Blot

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Mutation analysis of the EP domain within mNotch-1-IC. (A) Schematic representation of the mNotch-1-IC specific scanning mutants used in cotransfection experiments. (B) Transcriptional activity of EP domain mutants. Portions (2 μg) of reporter construct pGa981/6 were cotransfected into HeLa cells with 150-ng portions of plasmid expressing the indicated mNotch-1-IC proteins. Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. (C) Cell extracts from HEK-293 cells were prepared 24 h after transfection of 5 μg of plasmid expressing Notch-1-IC–EP domain mutant proteins. Expression was assayed by Western blotting using an anti-FLAG antibody. Mean values and standard deviations from four independent experiments are shown. (D) The EP domain is highly conserved within Notch proteins. Protein sequences of the indicated Notch proteins were aligned with the CLUSTAL W program. The first and last amino acids are numbered relative to the full-length proteins. The EP domain is specified. Abbreviations and accession numbers: N-dros, Notch Drosophila (P07207); N1-xen, Notch-1 Xenopus laevis (P21783); TAN-1, Notch-1 human (P46531); N1-mm, Notch-1 mouse (Q01705); N2-mm, Notch-2 mouse (BAA22094); N3-mm, Notch-3 mouse (Q61982).

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Mutagenesis, Cotransfection, Activity Assay, Construct, Plasmid Preparation, Expressing, Luciferase, Transfection, Western Blot

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Additive effect of p300 in Notch-1-mediated transactivation. Portions (2 μg) of reporter construct pGa981/6 were cotransfected into HeLa cells with 100 ng of plasmids expressing mNotch-1-IC (black bars) or the mNotch-1 specific EP domain mutant LDE/AAA (white bars), together with increasing amounts (50, 100, 200, and 500 ng) of pcDNA3-p300. Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and standard deviations of three independent experiments are shown.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Construct, Expressing, Mutagenesis, Luciferase, Activity Assay

Journal:

Article Title: p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

doi: 10.1128/MCB.21.22.7761-7774.2001

Figure Lengend Snippet: Transcriptional activation of the murine HES-1 promoter by Notch-1 depends on the EP domain. Portions (2 μg) of the HES-1 specific reporter construct HES-1-LUC were cotransfected into HeLa cells with 150 or 200 ng of plasmid expressing the indicated mNotch-1 proteins. Luciferase activity was determined from 100-μg portions of total-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and standard deviations from at least three independent experiments are shown.

Article Snippet: After blocking, the sections were incubated with the corresponding primary antibodies, anti-p300 (mouse monoclonal IgG [Upstate]) or anti-Notch-1 (M20, goat polyclonal IgG [Santa Cruz]) at 4°C overnight.

Techniques: Activation Assay, Construct, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Diagram of predicted O-glycans on mouse NOTCH1: red triangle, O-fucose glycans; blue circle, O-glucose glycans; blue square, O-GlcNAc glycans. Individual sugar residues that may extend O-fucose, O-glucose or O-GlcNAc to varying degrees are: yellow circle, galactose; pink diamond, sialic acid; orange star, xylose. ( B ) Flow cytometry of Lec1 CHO cells expressing vector control or Eogt siRNA with NOTCH1 mAb, DLL1-Fc, DLL4-Fc or JAG1-Fc. ( C ) Relative mean fluorescence index (MFI) for binding of DLL1-Fc and DLL4-Fc to control and Eogt -siRNA Lec1 cells. Concentrations of ligand varied from 100 to 750 ng/ml. MFI values for binding to control cells taken as 1.0 were 455 ± 55 (DLL1-Fc) and 1215 ± 49 (DLL4-Fc). Data from 9 to 10 independent experiments are average, normalized MFI ± SEM; significance determined by paired, two-tailed Student’s t-test, *p<0.05, ****p<0.0001. ( D ) MFI values obtained for binding of DLL1-Fc or DLL4-Fc (750 ng/ml) to control and Eogt knockdown Lec1 CHO cells, before and after transfection of a human EOGT cDNA. Data are mean ± SEM from three independent experiments. Significance determined by unpaired, two-tailed Student’s t-test, *p<0.05. Western blot analysis of transfectants. ( E ) DLL4 and JAG1 beads bound to wild-type, EOGT -null, or NOTCH1 -null HEK293T cells were observed by microscopy and counted (n = 50). Data are mean ± S.D. from three independent experiments. Statistical analysis was by Welch's t-test. **p<0.01; ***p<0.001. ( F ) Wild-type or EOGT -null HEK293T cells were transfected with Notch1 alone or together with Eogt followed by incubation with DLL4 beads. The number of DLL4 beads bound to cells was markedly increased by co-transfection of Eogt and Notch1. ( G ) Wild-type or EOGT -null HEK293T cells or cells transiently transfected with Notch1 with or without Eogt , were incubated with DLL4 or JAG1 beads. The number of Dynabeads bound per transfected cell marked by GFP expression was determined (n = 50). Data are mean ± S.D. from three independent experiments. *p<0.05; **p<0.01; ***p<0.001; # p<0.05; ### p<0.001 compared with the left-most wild type (*) or EOGT null (#) bar by Welch's t test. ( H ) Wild-type or EOGT -null HEK293T cells were transfected with Notch1 with or without Eogt and subjected to flow cytometry using 8G10 NOTCH1 Ab. Mock transfectants were analyzed with ( Mock ) or without primary antibody ( Cont ). DOI: http://dx.doi.org/10.7554/eLife.24419.003 10.7554/eLife.24419.004 Figure 1—source data 1. Raw data for . DOI: http://dx.doi.org/10.7554/eLife.24419.004

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, Control, Fluorescence, Binding Assay, Two Tailed Test, Knockdown, Transfection, Western Blot, Microscopy, Incubation, Cotransfection

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Schematic diagram of the CRISPR/Cas9 genome editing strategy to generate EOGT -null HEK293T cells. CRISPR/Cas9-mediated DNA cleavage caused frameshift mutations in all three EOGT alleles in HEK293T cells. ( B ) Wild type or EOGT -null HEK293T cells with Notch1-EGF-mycHis alone or together with EOGT . CTD110.6 immunoblotting revealed the lack of O-GlcNAc on NOTCH1 EGF repeats in the absence of EOGT. ( C ) Total ER fraction was obtained using Endoplasmic Reticulum Enrichment Extraction Kit (Novus Biologicals [NBP2-29482]) from parental HEK293T cells or EOGT -null cells, and subjected to immunoblotting with EOGT (1:2000 dilution) or BiP (1:10000 dilution) antibodies. ( D ) Schematic diagram of the CRISPR/Cas9 genome editing strategy to generate NOTCH1 -null HEK293T cells. CRISPR/Cas9-mediated DNA cleavage caused frameshift mutations in the single NOTCH1 allele in HEK293T cells. ( E ) Screening for CRISPR/Cas9-mediated genomic deletion at the Notch1 locus. A clone 1g1 was selected and deletion of the target sequence was confirmed by direct sequencing analysis. ( F ) Total cell lysates from parental HEK293T cells or NOTCH1 -null cells were subjected to immunoprecipitation (IP) using NOTCH1 ECD antibody (H-131). The immunoprecipitates were analyzed by immunoblotting with NOTCH1 ICD antibody (D6F11). Aliquots of total cell lysates were immunoblotted with β-tubulin antibody. DOI: http://dx.doi.org/10.7554/eLife.24419.005

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: CRISPR, Western Blot, Extraction, Sequencing, Immunoprecipitation

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Schematic of the EGF repeats of mammalian NOTCH1 identifying the potential O-GlcNAc consensus site, C 5 XXG(Y/F)(T/S)GXXC 6 , on EGF2, 10, 11, 14, 15, 17, 20, 23, 26, 27 and 29. These consensus sites are partly conserved in zebrafish and Drosophila . Recent studies of Drosophila Notch identified EGF4, 11, 12, 14 and 20 as major O-GlcNAcylated sites . ( B ) Schematic of EGF repeats of mouse Notch1 Δ2xO-GlcNAc and Notch1 Δ4xO-GlcNAc . DOI: http://dx.doi.org/10.7554/eLife.24419.008

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques:

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Ala substitution of Thr/Ser in the O-GlcNAc consensus site C 5 XXG(Y/F)(T/S)GXXC 6 in EGF2, 10, 17, and 20 in the NOTCH1 Δ4xO-GlcNAc mutant. ( B ) Cell surface expression of NOTCH1 Δ4xO-GlcNAc is comparable to that of wild-type NOTCH1. EOGT -null transfectants expressing Notch1 or Notch1 Δ4xO-GlcNAc with or without Eogt were subjected to flow cytometry using 8G10 NOTCH1 antibody. Control transfectants were with ( Mock ) or without ( Cont ) primary antibody. Similar results were obtained for wild-type HEK293T cells (not shown). ( C ) Expression and O-GlcNAcylation of Notch1 , Notch1 Δ2xO-GlcNAc and Notch1 Δ4xO-GlcNAc were analyzed by immunoprecipitation (IP) using NOTCH1 (8G10) antibody, followed by immunoblotting with CTD110.6 O-GlcNAc or NOTCH1 antibodies. ( D ) HEK293T transfectants expressing NOTCH1, NOTCH1 Δ2xO-GlcNAc or NOTCH1 Δ4xO-GlcNAc were immunostained for O-GlcNAc (CTD110.6 mAb; red) or NOTCH1 (8G10 mAb; green). Merged images include DAPI (blue) staining. Note that CTD110.6 mAb binding is markedly decreased in the NOTCH1 Δ4xO-GlcNAc mutant. ( E ) Quantification of the number of DLL4- or JAG1-coated Dynabeads bound to Notch1 versus Notch1 Δ4xO-GlcNAc transfected cells (marked with GFP), in the presence and absence of Eogt. Data are mean ± S.D from three independent experiments. Each experiment analyzed 50 cells. *p<0.05; **p<0.01; ***p<0.001 (Welch's t test). DOI: http://dx.doi.org/10.7554/eLife.24419.006 10.7554/eLife.24419.007 Figure 2—source data 1. Raw data for . DOI: http://dx.doi.org/10.7554/eLife.24419.007

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Mutagenesis, Expressing, Flow Cytometry, Control, Immunoprecipitation, Western Blot, Staining, Binding Assay, Transfection

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Knockdown of EOGT inhibits NOTCH1 activation cleavage. HeLa cells stably expressing shRNAs targeting GAPDH or EOGT (TA195, TA196, TA197, TA198) were co-cultured with L cells or D1/L (D1) cells in the presence and absence of 1 µM DAPT (GSI). After 6 hr, lysates were subjected to Western blot analysis using Abs to detect activated NOTCH1 (N1-act) and NOTCH1 full length (N1–FL) on the relevant section of the PVDF membrane. ( B ) Western blot analysis of samples from ( A ) using Ab to detect O-GlcNAc, followed after stripping by Ab to detect N1-FL. ( C ) HeLa cells stably expressing shRNA against GAPDH or EOGT (TA197) were transfected with vector control or a human EOGT cDNA. After 4 days, co-culture was performed with D1/L (D1) cells in the presence and absence of the DAPT. After ~7 hr, lysates were subjected to Western analysis to detect N1-FL, N1-act and EOGT on the relevant section of the PVDF membrane. O-GlcNAc was detected after stripping the N1-FL membrane section. * non-specific band. ( D ) The same as ( C ) except co-culture was with L cells or D1/L cells (D1). The second lane (#) was left empty. * non-specific band. ( E ) Knockdown of Eogt reduces ligand-induced Notch signaling. Lec1 CHO cells stably expressing siRNAs targeted against Eogt were transfected with TP1-luciferase and TK-renilla luciferase, co-cultured for 30 hr with L cells or D1/L cells, with and without GSI IX (12.5 μM) or a human EOGT cDNA, and dual firefly and renilla luciferase assays were performed. Normalized firefly luciferase activity in L versus D1/L cell co-cultures was plotted as fold-change. The number of independent experiments, each performed in duplicate, are shown in the histogram. Error bars are mean ± standard error and p values were determined by two-tailed paired Student’s t-test (****p < 0.0001). DOI: http://dx.doi.org/10.7554/eLife.24419.009 10.7554/eLife.24419.010 Figure 3—source data 1. Raw data for . DOI: http://dx.doi.org/10.7554/eLife.24419.010

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Knockdown, Activation Assay, Stable Transfection, Expressing, Cell Culture, Western Blot, Membrane, Stripping Membranes, shRNA, Transfection, Plasmid Preparation, Control, Co-Culture Assay, Luciferase, Activity Assay, Two Tailed Test

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Images of vessel density in P5 retinas from Eogt −/− compared to compound mutant mice. ( B ) Scatter plots represent branch point numbers in P5 retinas from Eogt −/− compared with compound mutant mice, normalized to Eogt +/+ mice. The average number of branch points for the Eogt +/+ used to compare mice with an N1 − or Rbpj − allele was 237 ± 5, and for the Eogt +/+ compared to mice with an N 12f or N1 lbd allele was 380 ± 36 (500 X 500 μm field (n = 3–8 fields per retina), N = 2–6 mice). ( C ) Scatter plots showing the number of filopodia in P5 compound mutant mice as compared to Eogt −/− mice, normalized to Eogt +/+ mice. Each symbol represents the average number of filopodia per mouse (250 × 250 µm field (n = 4–12 fields per mouse), N = 2–4 mice. The average for Eogt +/+ compared to mice with a N1 - or Rbpj - allele was 30 ± 1 per mm and for mice with a N1 12f or N1 lbd allele was 36 ± 4 per mm, taken as 1.0 for normalization. ( D ) Images of branch points in P15 retinas comparing Eogt −/− to compound mutant mice as indicated. ( E ) Scatter plots shows branch points in compound mutants compared to Eogt −/− P15 retinas, normalized to Eogt +/+ mice. The average of the Eogt +/+ used to compare mice with a with a N1 − or Rbpj − allele was 73 ± 3, and for mice with a N1 12f or N1 lbd allele it was 69 ± 12 (500 x 500 µm field (n = 3–8 fields per retina), N = 3–6 mice. Data represent mean ± standard error except for P5 Eogt +/+ Notch1 +/− filopodia and P5 Eogt +/+ Notch1 +/lbd branch points and filopodia that are represented as mean ± range; p values were calculated by unpaired two-tailed Student’s t-test. *p≤0.05; **p≤0.01; ***p≤0.001. DOI: http://dx.doi.org/10.7554/eLife.24419.017

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Mutagenesis, Two Tailed Test

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: ( A ) Immunostaining with fibrinogen (green) and α-SMA (magenta) antibodies in P15 wild-type, Eogt −/− , Tek-Cre , Tek-Cre:Eogt F/F , Notch1 +/− , and Rbpj +/− retinas. Arrows indicate fibrinogen staining outside vessels stained by IB4 (white). Three-dimensional images were constructed from confocal images by maximum intensity projection. ( B ) Higher magnification three-dimensional images of Eogt −/− retina constructed from confocal images using the Alpha-blend method. Below , single channel images showing fibrinogen (green) and IB4 (white) staining. ( C ) Sulfo-NHS-LC-biotin was perfused into P15 wild-type and Eogt −/− mice and extravasation determined immediately after perfusion by staining with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white). Three-dimensional reconstructions were created by maximum intensity projection. Enlarged images of boxed area are shown (right). ( D ) Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt −/− , Notch1 +/− , Eogt −/− Notch1 +/− mice as in ( C ). Quantification of the number of extravasation sites in 210 × 210 μm squares (n = 6 per retina per mouse) is shown. Note that sulfo-NHS-LC-biotin extravasation in Eogt −/− retina is augmented in compound mutant mice. Data represent mean ± standard error; p values determined by Welch's t test. ***p≤0.001. ( E ) Whole-mount images of wild-type or Eogt −/− P15 retinas stained with IB4 (cyan) and anti-αSMA (magenta) antibody. ( F ) Whole-mount staining of wild-type and Eogt −/− P15 retinas using IB4 (white) together with anti-fibrinogen (green) and anti-NG2 (magenta) antibodies. DOI: http://dx.doi.org/10.7554/eLife.24419.018 10.7554/eLife.24419.019 Figure 7—source data 1. Raw data for . DOI: http://dx.doi.org/10.7554/eLife.24419.019

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Immunostaining, Staining, Construct, Mutagenesis

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/eLife.24419

Figure Lengend Snippet: Primers used for qRT-PCR. DOI: http://dx.doi.org/10.7554/eLife.24419.025

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–α-smooth muscle actin (αSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-αSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) , hamster anti-mouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) , sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRP-conjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: