Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 1. Morphological examination of caveolae in resting EA.hy926 cells. A: Transmission electron microscopy reveals caveolae, indicated by arrow- heads, in resting EA.hy926 cells. B: EM analysis of MCD-treated cells showing the disappearance of caveolae network. C: Confocal immunofluo- rescence microscopy with a combination of anti-TNFR1/caveolin antibodies in both control (top) and MCD-treated (bottom) EA.hy926 cells. White boxes indicate high magnification (right). Note co-localization of TNFR1 and caveolin in the periphery of control but not MCD-treated cells. Data are from two different independent experiments.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Transmission Assay, Electron Microscopy, Microscopy, Control

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 2. Fractionation of EA.hy926 cells on sucrose gradient. A: EA.hy926 cells were harvested and fractionated by sucrose density gradient as de- scribed in Materials and Methods and 10 l of each sample was subjected to a dot-blot analysis by staining membrane with HRP-conjugated cholera toxin subunit B (CTxB). B: EA.hy926 cells were incubated for 30 minutes with two different doses of methyl--cyclodextrin (MCD) or not treated, Dounce homogenized, and then fractionated by sucrose density gradient. Fractions, harvested from the top to the bottom of each gradient were analyzed by SDS-PAGE and immunoblotted for TNFR1 and caveolin-1. Ctr, indicates control cells not subjected to MCD treatment. C: Fractions 3 to 5, pooled after fractionation of resting cells, were subjected to immunoelectron micros- copy analysis with both TNFR1 (15 nm, arrowhead) and caveolin (5 nm, arrow) antibodies. Note co-localization of TNFR1 and caveolae in isolated caveolae.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Fractionation, Dot Blot, Staining, Membrane, Incubation, SDS Page, Control, Isolation

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 5. Co-immunoprecipitation of TNFR1 and caveolin-1 from caveolae in EA.hy926 cells. EA.hy926 cells were stimulated with TNF for the indicated time and fractionated by sucrose gradient as described in Materials and Methods. Four hundred l from fractions 3, 4, and 5 (left), and from 9, 10, and 11 were mixed and immunoprecipitated with TNFR1 followed by SDS- PAGE analysis with both TNFR1 and caveolin-1 antibodies. Note that al- though both the proteins are represented in fractions 9 to 11 (see Figure 4A), no interaction between the two proteins was detected. Immunoprecipitation of TNFR1 performed in resting cells not subjected to the fractionation pro- cedure, shows the ability of MCD to disrupt the association between TNFR1 and caveolin-1, as indicated by R at the left. Data are from one of three independent experiments with similar results.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Immunoprecipitation, SDS Page, Fractionation

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 6. TNFR1 does not internalize in clathrin-coated structures in EA.hy926 cells. A: EA.hy926 cells were either left untreated or treated for different periods of time with 10 ng/ml of TNF as indicated and analyzed by laser confocal microscopy after immunostaining with a combination of goat anti-human TNFR1 and mouse anti-clathrin heavy chain antibodies. B: Con- focal analysis of clathrin and transferrin receptor (TfR) in resting EA.hy926 cells. Right: Higher magnification (indicated by box) of the overlay image. Images shown are representative of three independent experiments with similar results. Original magnifications, 63.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Confocal Microscopy, Immunostaining

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 7. TNF-induces trafficking of TNFR1 to endosomes in EA.hy926 cells. EA.hy926 cells were treated with 10 ng/ml of TNF and subjected to confocal fluorescent microscopy by staining with a combination of TNFR1 with either EEA1 (A) or Rab5 (B) antibodies. Images shown are from one of three different experiments with similar results.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Microscopy, Staining

Journal:

Article Title: Tumor necrosis factor-related apoptosis-inducing ligand induces neuronal death in a murine model of HIV central nervous system infection

doi: 10.1073/pnas.2628048100

Figure Lengend Snippet: HIV-CNS infection and apoptosis in HIV-infected hu-PBMC-NOD-SCID mice

Article Snippet: Frozen sections were incubated with Abs against human CD68 (clone PGM1), human TRAIL (clone RIK-2), IL-1β (rabbit polyclonal IgG, Endogen, Woburn, MA), human TNF-α (rabbit polyclonal IgG, Endogen), human FasL (rabbit polyclonal IgG, Santa Cruz Biotechnology), HIV-1 gag p24 (goat polyclonal IgG, ViroStat, Portland, ME), or the active form of caspase-3 (rabbit polyclonal IgG, R & D Systems).

Techniques: Infection, Virus, TUNEL Assay

Journal:

Article Title: Tumor necrosis factor-related apoptosis-inducing ligand induces neuronal death in a murine model of HIV central nervous system infection

doi: 10.1073/pnas.2628048100

Figure Lengend Snippet: Association of apoptotic cells with HIV-1-infected macrophages expressing death-inducing molecules. (a) Expression of TRAIL, TNF-α, or FasL on HIV-1-infected macrophage. hu-PBMC-NOD-SCID mice were infected with M-tropic NL-CSFV3-EGFP and LPS was given or not 7 days after infection. Three days later, brain sections were subjected to histological analysis for GFP (green), human CD68 (red), and human TRAIL, TNF-α, or FasL (blue) simultaneously. A representative cell is shown. (Scale bars, 10 μm.) (b) hu-PBMC-NOD-SCID mice were infected with JRFL, and LPS was given 7 days after infection. Three days later, brain sections were subjected to dual-color staining for human CD68, HIV gag p24, or human TRAIL (green) and TUNEL (red). (Scale bars, 100 μm.)

Article Snippet: Frozen sections were incubated with Abs against human CD68 (clone PGM1), human TRAIL (clone RIK-2), IL-1β (rabbit polyclonal IgG, Endogen, Woburn, MA), human TNF-α (rabbit polyclonal IgG, Endogen), human FasL (rabbit polyclonal IgG, Santa Cruz Biotechnology), HIV-1 gag p24 (goat polyclonal IgG, ViroStat, Portland, ME), or the active form of caspase-3 (rabbit polyclonal IgG, R & D Systems).

Techniques: Infection, Expressing, Staining, TUNEL Assay