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: Virulence

Article Title: Mucorales spores induce a proinflammatory cytokine response in human mononuclear phagocytes and harbor no rodlet hydrophobins

doi: 10.1080/21505594.2017.1342920

Figure Lengend Snippet: Dormant R. arrhizus spores induce the upregulation of co-stimulatory molecules on moDCs (A) Geometric mean fluorescence values for CD83 and CD86 expression after an 18 h co-culture of 5 × 105 moDCs with 5 × 105 spores (Sp) and germ tubes (GT) of A. fumigatus (Afu) or R. arrhizus (Rar). (B) Gating strategy and CD83/CD86 plots for moDCs from one representative donor. CD1+ CD14− cells were identified among all harvested cells. Within this subset, geometric mean fluorescence for CD83-PE and CD86-APC was quantified.

Article Snippet: 300 µl (3 × 10 5 cells) were plated in each well of a 48-well plate and co-incubated with ethanol-inactivated fungal spores or germ tubes for 18 h. The cells were then transferred to FACS tubes, washed and stained in 100 µl HBSS containing CD1a-FITC, CD14-PerCP, CD83-PE, and CD86-APC antibodies (Miltenyi Biotec, #130–097–903, #130–094–969, #130–094,876; BD Biosciences, #556855).

Techniques: Fluorescence, Expressing, Co-Culture Assay

Journal: Heliyon

Article Title: Abcam Monoclonal Egr-1 ab133695 is an effective primary antibody replacement for Santa Cruz sc-189 polyclonal Egr-1 in songbirds ☆

doi: 10.1016/j.heliyon.2019.e02938

Figure Lengend Snippet: Effectiveness of primary antibody protocol. IEG labeling in the CMM of a song-exposed male zebra finch for each treatment; (A) 1:1000 Santa Cruz erg-1 1-day incubation, (B) 1:1000 Abcam Egr-1 ab133695 1-day incubation, (C) 1:1000 Abcam Egr-1 2-day incubation, (D) 1:2000 Abcam Egr-1 1-day incubation, (E) 1:2000 Abcam Egr-1 2-day incubation, (F) 1:5000 Abcam Egr-1 1-day incubation, (G) 1:5000 Abcam Egr-1 2-day incubation, (H) 1:1000 Proteintech Egr-1 55117-1-AP 1-day incubation, (I) 1:1000 Proteintech Egr-1 2-day incubation, (J) 1:500 Proteintech Egr-1 1-day incubation, (K) 1:1000 Abcam c-Fos ab209794 2-day incubation, (L) 1:500 Abcam c-Fos 2-day incubation (M) Scaled proportion (scaled to the highest overall count) of IEG marked cells per treatment in silence and song-exposed zebra finch males. Counts from (B) and (C) were not included in the graph (M) as the tissue was burned during the immunohistochemistry procedure rendering them unscorable. Scale bar = 50 μm, same for all images.

Article Snippet: A) 1:1000 Santa Cruz Erg-1 sc-189 1-day incubation (Santa Cruz Biotechnology, Santa Cruz, CA, USA), B) 1:1000 Abcam Egr-1 ab133695 1-day incubation (Abcam Inc, Toronto, ON, Canada, C) 1:1000 Abcam Egr-1 ab133695 2-day incubation, D) 1:2000 Abcam Egr-1 ab133695 1-day incubation, E) 1:2000 Abcam Egr-1 ab133695 2-day incubation, F) 1:5000 Abcam Egr-1 ab133695 1-day incubation, G) 1:5000 Abcam Egr-1 ab133695 2-day incubation, H) 1:1000 Proteintech Egr-1 55117-1-AP 1-day incubation (Proteintech, Rosemount, IL, USA), I) 1:1000 Proteintech Egr-1 55117-1-AP 2-day incubation, J) 1:500 Proteintech Egr-1 55117-1-AP 1-day incubation, K) 1:1000 Abcam c-Fos ab209794 2-day incubation, L) 1:500 Abcam c-Fos ab209794 2-day incubation.

Techniques: Labeling, Incubation, Immunohistochemistry