dr5  (R&D Systems)

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: (A) 293T cells were transfected with a cDNA plasmid encoding Strep-DR5 fusion protein or an empty vector as a control. Shown are representative DR5 immunoblots. (B) Strep-DR5 protein complexes were isolated using Strep-Tactin affinity chromatography and resolved onto SDS-PAGE followed by Coomassie blue staining. Labeled bands were identified by mass spectrometry as tubulin (band 1) and DR5 (band 2). (C) Anti-DR5 immunoprecipitation followed by SDS-PAGE and silver staining. Labeled bands are as follows: 1, Polyubiquitin-B/C; 2, Isoleucine-tRNA ligase; 3, Exportin-2; 4, HSP 90-beta; 5, Tubulin & DR5. * unknown component in the commercial anti-DR5 antibody, ** IgG heavy chain.

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Isolation, Affinity Chromatography, SDS Page, Staining, Labeling, Mass Spectrometry, Immunoprecipitation, Silver Staining

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: (A) Immunoblots of endogenous DR5 and β-tubulin proteins in MB-231 and H460 cell lines. (B) Cell lysates were subjected to IP with anti-DR5 antibodies or IgG1 or IgG2b control s . IP samples were immunoblotted as indicated. (C) Reverse co-IP using anti-tubulin antibodies confirmed the interaction between tubulin and DR5.

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Control, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: (A) Cells were treated with colchicine (tubulin inhibitor) at 100 ng/ml for or (B) taxol (tubulin stabilizer) at 50 nM 16 h, and analyzed by immunoblotting or (C) flow cytometry. DR5 total expression is relative to untreated controls. Values are means ± SD of triplicates, * p<0.05, ** p<0.01, N=3.

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Flow Cytometry, Expressing

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: (A) Cells were treated with colchicine at 100 ng/mL or taxol at 50 nM for 16 hours and then cell lysates were subjected to IP with anti-DR5 antibodies or IgG2b control. (B) MB-231 cells were treated with colchicine at 50 ng/mL or taxol at 25 nM for 4 hours and stained for α-tubulin (green) and DR5 (red) with DAPI co-staining. Cells were visualized using confocal microscopy. Yellow arrows indicate co-localization of α-tubulin and DR5. Images were taken at 40x magnification.

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Control, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: (A) Cells were transfected with control siRNA (-) or siRNA against α-tubulin (+) for 48 hr, followed by immunoblot analysis. (B) Relative levels of cell surface DR5 was determined by flow cytometry. Cells were prepared as in (A) and stained with PE-conjugated monoclonal anti-DR5 antibody or isotype-matched control IgG (IgG2b). Data shown is relative to IgG2b-PE isotype controls. Values are means ± SD, ** p<0.01, N=3. (C & D) Cells were transfected with siRNA as in (A), treated with TRAIL at 10 ng/ml for 24 h, followed by analyses for cell viability (C) and caspase activation (D).

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control, Western Blot, Flow Cytometry, Staining, Activation Assay

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: (A) MDA-MB-231 cells were left untreated or treated with 500 ng/ml of colchicine followed by stimulation with 20 μg/ml of cycloheximide (CHX; a protein synthesis inhibitor) for the indicated times. Right panel, relative DR5 protein levels were quantified from the blots of DR5 and corresponding GAPDH in each sample. (B) MDA-MB-231 cells were transfected with control siRNA or siRNA against tubulin for 48 h, and were then treated with 20 μg/ml of CHX for the indicated times, and analyzed as in A. Values are means ± SD, * p<0.05, N=3.

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control

Journal: Oncotarget

Article Title: Tubulin couples death receptor 5 to regulate apoptosis

doi: 10.18632/oncotarget.26407

Figure Lengend Snippet: Tubulin and DR5 are found to form stable complexes which appears to promote DR5 protein degradation via undefined pathways. Pharmacological disruption of tubulin network using either depolymerizing (e.g., colchicine) or stabilizing (e.g. taxol) agent increases DR5 total and surface expression. In both cases, the resultant cells are sensitized to TRAIL induced apoptosis. Our data suggest distinct mechanisms: 1) Tubulin depolymerization appears to lower affinity to DR5 which results in release and stabilization of DR5 protein, and 2) Tubulin stabilization shows little or effect on DR5-tubulin complex formation, but it may act via inducing DR5 transcriptional expression . In either case, the upregulated DR5 protein levels may be responsible for the increased surface expression and subsequent apoptosis in response to TRAIL treatment.

Article Snippet: Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems (Minneapolis, MN).

Techniques: Disruption, Expressing