Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 2. Characterization of the signaling capacity of Jagged1WT, Jagged1Ndr, Jagged1Slm and Jagged1Htu. (A)12XCSL-luciferease reporter activity in co- cultures of Notch-expressing cells (carrying 12XCSL-luc) and cells expressing wild-type or mutated Jagged1. Bar graphs show luciferase activity (arithmetic mean) from triplicates of each co-culture. Error bars indicate s.d.; ***P<0.001. (B)Western blot analysis of lysates from stable cell lines (Flp-in HEK293 cells) expressing no Jagged1 (–), Jagged1WT, Jagged1Ndr, Jagged1Slm, and Jagged1Htu show comparable levels of Jagged1 (reprobed with -actin as loading control). (C)Cell lines expressing Jagged1WT, but not Jagged1Ndr, Jagged1Slm and Jagged1Htu, bind N1ECD-Fc-Cy3 as assessed by FACS analysis of cells exposed to N1ECD-Fc-Cy3 for 1 hour. (D)Cells expressing Jagged1WT, but not Jagged1Ndr, Jagged1Slm and Jagged1Htu, bind N1ECD-Fc-Cy3 (red), whereas all four cell lines express Jagged1 (green) at the cell surface. Scale bar: 20m. (E)Streptavidin-precipitation of cell-surface biotinylated proteins (top panel; without biotin as control, bottom panel) from HEK293 cells stably expressing Jagged1WT, Jagged1Ndr, Jagged1Slm and Jagged1Htu. Reprobing for nicastrin shows that only the mature, cell-surface-bound form, is detected in the streptavidin-precipitated material (the five lanes to the right). (F)12XCSL-luciferease reporter activity from Notch-expressing cells (carrying 12XCSL-luc) co-cultured with cells expressing wild-type Jagged1 and increasing levels of Jagged1Ndr. No dominant-negative effect by Jagged1Ndr is observed.

Article Snippet: The following primary and secondary antibodies were used in the study: rabbit antiJagged1 (Santa Cruz), goat anti-Jagged1 (Santa Cruz), rabbit anti-pan Cadherin (BD Biosciences), mouse anti-HA (Nordic Biosite and Covance), rabbit anti-GFP (Abcam), rat anti-PECAM (BD Biosciences), rabbit anti-nicastrin (Sigma), anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen) anti-mouse Cy3 (Jackson), anti-mouse Cy5 (Jackson), anti-rabbit Alexa Fluor 488 (Molecular Probes/Invitrogen), anti-goat Alexa Fluor 488 (Molecular probes/Invitrogen), anti-goat Cy3 (Jackson) anti-human Cy3 (Jackson), anti-human Cy5 (Jackson), anti-rat HRP (Vector Laboratories), antigoat HRP (DAKO), anti-mouse HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques: Activity Assay, Expressing, Luciferase, Co-Culture Assay, Western Blot, Stable Transfection, Control, Cell Culture, Dominant Negative Mutation

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 3. Mib1 and endocytosis of Jagged1 are required for activation of, but not binding to, Notch receptors. (A,B)12XCSL-luciferease reporter activity in full-length Notch1-expressing cells co-cultured with cells transiently transfected with (A) EGFP, Jagged1+EGFP or Jagged1+EGFP-tagged DynaminII K44A as indicated or (B) control siRNA, Jagged1+control siRNA or Jagged1+siRNA specific for MIB1 as indicated. Bar graphs show luciferase activity (arithmetic mean) from triplicates of each co-culture. Luciferase activity was normalized to control cultures transfected with EGFP (A) or control siRNA (B). Error bars indicate s.d.; **P<0.01. (C)Immunocytochemistry demonstrating that cells transfected with DynaminII K44A and Jagged1 (top cell) can bind N1ECD-Fc-Cy3, whereas cells not transfected with DynaminII K44A and Jagged1 (the bottom cell) does not bind N1ECD-Fc-Cy3. As a control for block of endocytosis by DynaminII K44A, there is no transferrin uptake in the top (DynaminII K44A expressing) cell, whereas transferrin is internalized in the bottom cell. Scale bar: 10m. Immunofluorescence images were adjusted using the levels function in Photoshop. In each case, adjustments were applied to the entire image. (D)Detection of ligand-receptor interaction by flow cytometry in cells transiently transfected with empty plasmid and EGFP, Jagged1 and EGFP or DynaminII K44A and EGFP, as indicated. (E)Detection of ligand-receptor interaction by flow cytometry in cells transiently transfected with empty plasmid and control siRNA, Jagged1 and control siRNA or Jagged1 and MIB1 siRNA, as indicated. Percentages of fluorescently labeled cells (arithmetic mean) is shown (bar graphs). Error bars indicate s.d.; *P<0.05, **P<0.01.

Article Snippet: The following primary and secondary antibodies were used in the study: rabbit antiJagged1 (Santa Cruz), goat anti-Jagged1 (Santa Cruz), rabbit anti-pan Cadherin (BD Biosciences), mouse anti-HA (Nordic Biosite and Covance), rabbit anti-GFP (Abcam), rat anti-PECAM (BD Biosciences), rabbit anti-nicastrin (Sigma), anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen) anti-mouse Cy3 (Jackson), anti-mouse Cy5 (Jackson), anti-rabbit Alexa Fluor 488 (Molecular Probes/Invitrogen), anti-goat Alexa Fluor 488 (Molecular probes/Invitrogen), anti-goat Cy3 (Jackson) anti-human Cy3 (Jackson), anti-human Cy5 (Jackson), anti-rat HRP (Vector Laboratories), antigoat HRP (DAKO), anti-mouse HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques: Activation Assay, Binding Assay, Activity Assay, Expressing, Cell Culture, Transfection, Control, Luciferase, Co-Culture Assay, Immunocytochemistry, Blocking Assay, Immunofluorescence, Flow Cytometry, Plasmid Preparation, Labeling

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 7. NECD is transendocytosed into Jagged1WT-expressing cells and transported through the endocytic degradation pathway. (A)HEK293T cells expressing full length HA-Notch1 (N-terminally tagged Notch1) were co-cultured with Jagged1WT-expressing HEK293T cells transfected with EGFP-EEA1. HA immunoreactivity was observed in EGFP-positive and Jagged1-positive vesicles indicating trans-endocytosis of Notch1 ECD into EEA1-positive vesicles in Jagged1-expressing cells. Boxed region is magnified in inset. Scale bar: 20m. (B)Jagged1WT cells exhibit high levels of trans-endocytosis of Notch1 ECD, whereas Jagged1Ndr cells exhibit low-to-no trans-endocytosis of Notch1ECD into Jagged1- and EEA1-positive vesicles (arrowheads). Scale bar: 20m. (C-E)Immunocytochemistry of cells with fluorescently labeled subcellular markers and incubated with N1ECD-Fc-Cy3. (C)Immediately after incubation, N1ECD-Fc-Cy3 (red) colocalized with the plasma-membrane marker EGFP-F. (D)At 1 hour, N1ECD-Fc-Cy3 (red) was detected in EGFP-EEA1-positive vesicles. (E)At 4 hours, N1ECD-Fc-Cy5 (green) was detected in lysosomes, as visualized by the lysosome marker Lysotracker Red (red).

Article Snippet: The following primary and secondary antibodies were used in the study: rabbit antiJagged1 (Santa Cruz), goat anti-Jagged1 (Santa Cruz), rabbit anti-pan Cadherin (BD Biosciences), mouse anti-HA (Nordic Biosite and Covance), rabbit anti-GFP (Abcam), rat anti-PECAM (BD Biosciences), rabbit anti-nicastrin (Sigma), anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen) anti-mouse Cy3 (Jackson), anti-mouse Cy5 (Jackson), anti-rabbit Alexa Fluor 488 (Molecular Probes/Invitrogen), anti-goat Alexa Fluor 488 (Molecular probes/Invitrogen), anti-goat Cy3 (Jackson) anti-human Cy3 (Jackson), anti-human Cy5 (Jackson), anti-rat HRP (Vector Laboratories), antigoat HRP (DAKO), anti-mouse HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques: Expressing, Cell Culture, Transfection, Immunocytochemistry, Labeling, Incubation, Clinical Proteomics, Membrane, Marker