mouse anti tgn46 Search Results


tgn46  (Bio-Rad)
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Bio-Rad tgn46
Depletion of ORP5 impairs the transport of CI-MPR and <t>TGN46,</t> but not STxB. (A) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by double-labeling with antibodies to CI-MPR and giantin. Confocal images are shown to indicate a more dispersed distribution of CI-MPR in cells treated with siORP5 rather than with siNPC1. Individual fluorescent profiles in merged images were quantified using the Olympus FluoView version 2.0 Viewer software and are shown on the right. (B) Quantification of cells with dispersed CI-MPR in A. Values are the percentage of cells with dispersed localization of CI-MPR (means + SD [error bars], n > 100; *, P < 0.05; **, P < 0.01). (C) HeLa cells were transfected with the indicated siRNA for 72 h, incubated on ice for 10 min, and chased with mouse monoclonal antibodies to CI-MPR for 30 min on ice. Antibody internalization was performed at 37°C for 60 min. Cells were then fixed by 4% paraformaldehyde, and internalized CI-MPR was detected using Alexa Fluor 488–conjugated goat anti–mouse antibodies. (D) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by labeling with antibodies to <t>TGN46.</t> Confocal images are shown to indicate a dispersed distribution of TGN46 in cells treated with siORP5. (E) Quantification of cells with dispersed TGN46 in D. Values are the percentage of cells with dispersed localization of TGN46 (means + SD [error bars], n > 100; **, P < 0.01). Data are representative of three independent experiments with similar results. (F) HeLa cells, transfected with the indicated siRNA for 72 h in medium A, were incubated with Cy3-conjugated STxB in DME, 1% BSA, and 25 mM Hepes, pH 7.4, for 15 min at 37°C. Cells were washed in PBS and incubated for 1 h at 37°C in medium A before fixation by 4% paraformaldehyde, followed by labeling with antibodies to giantin. Bars, 10 µm.
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Depletion of ORP5 impairs the transport of CI-MPR and TGN46, but not STxB. (A) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by double-labeling with antibodies to CI-MPR and giantin. Confocal images are shown to indicate a more dispersed distribution of CI-MPR in cells treated with siORP5 rather than with siNPC1. Individual fluorescent profiles in merged images were quantified using the Olympus FluoView version 2.0 Viewer software and are shown on the right. (B) Quantification of cells with dispersed CI-MPR in A. Values are the percentage of cells with dispersed localization of CI-MPR (means + SD [error bars], n > 100; *, P < 0.05; **, P < 0.01). (C) HeLa cells were transfected with the indicated siRNA for 72 h, incubated on ice for 10 min, and chased with mouse monoclonal antibodies to CI-MPR for 30 min on ice. Antibody internalization was performed at 37°C for 60 min. Cells were then fixed by 4% paraformaldehyde, and internalized CI-MPR was detected using Alexa Fluor 488–conjugated goat anti–mouse antibodies. (D) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by labeling with antibodies to TGN46. Confocal images are shown to indicate a dispersed distribution of TGN46 in cells treated with siORP5. (E) Quantification of cells with dispersed TGN46 in D. Values are the percentage of cells with dispersed localization of TGN46 (means + SD [error bars], n > 100; **, P < 0.01). Data are representative of three independent experiments with similar results. (F) HeLa cells, transfected with the indicated siRNA for 72 h in medium A, were incubated with Cy3-conjugated STxB in DME, 1% BSA, and 25 mM Hepes, pH 7.4, for 15 min at 37°C. Cells were washed in PBS and incubated for 1 h at 37°C in medium A before fixation by 4% paraformaldehyde, followed by labeling with antibodies to giantin. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking

doi: 10.1083/jcb.201004142

Figure Lengend Snippet: Depletion of ORP5 impairs the transport of CI-MPR and TGN46, but not STxB. (A) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by double-labeling with antibodies to CI-MPR and giantin. Confocal images are shown to indicate a more dispersed distribution of CI-MPR in cells treated with siORP5 rather than with siNPC1. Individual fluorescent profiles in merged images were quantified using the Olympus FluoView version 2.0 Viewer software and are shown on the right. (B) Quantification of cells with dispersed CI-MPR in A. Values are the percentage of cells with dispersed localization of CI-MPR (means + SD [error bars], n > 100; *, P < 0.05; **, P < 0.01). (C) HeLa cells were transfected with the indicated siRNA for 72 h, incubated on ice for 10 min, and chased with mouse monoclonal antibodies to CI-MPR for 30 min on ice. Antibody internalization was performed at 37°C for 60 min. Cells were then fixed by 4% paraformaldehyde, and internalized CI-MPR was detected using Alexa Fluor 488–conjugated goat anti–mouse antibodies. (D) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by labeling with antibodies to TGN46. Confocal images are shown to indicate a dispersed distribution of TGN46 in cells treated with siORP5. (E) Quantification of cells with dispersed TGN46 in D. Values are the percentage of cells with dispersed localization of TGN46 (means + SD [error bars], n > 100; **, P < 0.01). Data are representative of three independent experiments with similar results. (F) HeLa cells, transfected with the indicated siRNA for 72 h in medium A, were incubated with Cy3-conjugated STxB in DME, 1% BSA, and 25 mM Hepes, pH 7.4, for 15 min at 37°C. Cells were washed in PBS and incubated for 1 h at 37°C in medium A before fixation by 4% paraformaldehyde, followed by labeling with antibodies to giantin. Bars, 10 µm.

Article Snippet: Antibodies used were goat polyclonal to ORP5 (Abcam) and β-1,4-galactosyltransferase I (β-1,4-GT1; Santa Cruz Biotechnology, Inc.), rabbit polyclonal to the C-terminal region of human NPC1 and golgin 97 (Abcam), GFP (Invitrogen), syntaxin 5 , calnexin (Cell Signaling Technology), DsRed (Takara Bio, Inc.), giantin (Covance), and TGN46 (AbD Serotec), mouse monoclonal to the Xpress tag (Invitrogen), actin (Abcam), GST, Lamp-1 and GFP (Santa Cruz Biotechnology, Inc.), and GM130 (BD).

Techniques: Transfection, Labeling, Software, Incubation, Bioprocessing

Rescue effects of ORP5 expression. (A) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with EGFP alone or RNAi-resistant GFP-OPR5 (GFP-ORP5-siR) for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Images are shown to indicate that cholesterol accumulation was significantly alleviated in GFP-ORP5-siR–expressing cells (71 ± 2%) but not in GFP-expressing cells (5 ± 3%). (B) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant GFP-OPR5 for 24 h. Cells were fixed and labeled with antibodies to TGN46. Confocal images are shown to indicate that the dispersed distribution of TGN46 was rescued in cells expressing GFP-ORP5. (C) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant mCherry-OPR5 for 24 h. Cells were fixed and labeled with antibodies to CI-MPR. Confocal images are shown to indicate that the dispersed distribution of CI-MPR was rescued in cells expressing mCherry-ORP5. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking

doi: 10.1083/jcb.201004142

Figure Lengend Snippet: Rescue effects of ORP5 expression. (A) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with EGFP alone or RNAi-resistant GFP-OPR5 (GFP-ORP5-siR) for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Images are shown to indicate that cholesterol accumulation was significantly alleviated in GFP-ORP5-siR–expressing cells (71 ± 2%) but not in GFP-expressing cells (5 ± 3%). (B) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant GFP-OPR5 for 24 h. Cells were fixed and labeled with antibodies to TGN46. Confocal images are shown to indicate that the dispersed distribution of TGN46 was rescued in cells expressing GFP-ORP5. (C) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant mCherry-OPR5 for 24 h. Cells were fixed and labeled with antibodies to CI-MPR. Confocal images are shown to indicate that the dispersed distribution of CI-MPR was rescued in cells expressing mCherry-ORP5. Bars, 10 µm.

Article Snippet: Antibodies used were goat polyclonal to ORP5 (Abcam) and β-1,4-galactosyltransferase I (β-1,4-GT1; Santa Cruz Biotechnology, Inc.), rabbit polyclonal to the C-terminal region of human NPC1 and golgin 97 (Abcam), GFP (Invitrogen), syntaxin 5 , calnexin (Cell Signaling Technology), DsRed (Takara Bio, Inc.), giantin (Covance), and TGN46 (AbD Serotec), mouse monoclonal to the Xpress tag (Invitrogen), actin (Abcam), GST, Lamp-1 and GFP (Santa Cruz Biotechnology, Inc.), and GM130 (BD).

Techniques: Expressing, Transfection, Staining, Fluorescence, Microscopy, Labeling