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: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 5 | Interfering with EGR1 inhibited the proliferation and cycle acceleration of DLBCL cells and promotes apoptosis. The EGR1 gene in cells was silenced through siRNA transfection. (A) The interference level of EGR1 was measured by western blot analysis. (B) Cell proliferation was detected by CCK8 assay. (C) EdU staining was performed to evaluate the level of cell proliferation; Flow cytometry was conducted to analyze the cell cycle (D) and the level of apoptosis (E). (F) The expressions of CtBP2 and apoptosis‐related proteins were detected by western blot. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Transfection, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 6 | Interfere with CtBP2 inhibited the proliferation and cycle acceleration of DLBCL cells and promoted apoptosis through EGR1. The cells were transfected with an Ov‐EGR1 overexpression plasmid. (A) The overexpression level of EGR1 was assessed using Western blot analysis. (B) Cell proliferation was evaluated by CCK‐8 assay. (C) Cell proliferation was measured through EdU staining; Flow cytometry was performed to analyze the cell cycle (D) and the level of apoptosis (E). (F) The expression of apoptosis‐related proteins was detected by western blot assay. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, CCK-8 Assay, Staining, Flow Cytometry, Expressing

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 7 | EGR1 may be involved in the regulation of the Wnt/β‐catenin signaling pathway by CtBP2. The expressions of Wnt/β‐catenin signaling‐related proteins DKK1, β‐catenin and c‐MyC were detected by western blot. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

doi: 10.1038/s41467-019-13616-1

Figure Lengend Snippet: a SPR depicts the binding of UCB- 6786 to immobilised TNF. b The analogue, UCB- 6876 , specifically binds to immobilised TNF (solid black circles) but not to control proteins, capture antibody plus TNFR1 (solid black squares), capture antibody (solid black triangle pointed down) or TNFR1 (solid black triangle pointed up). c Kinetic analysis of UCB- 6876 using the Biacore T100 showing slow association and dissociation rates.

Article Snippet: TNFR1 immunoprecipitation was performed by incubating supernatant with 5 μg anti-TNFR1 antibody (R&D systems, #AB-225-PB) overnight while rotating at 4 °C, followed by the addition of 20 μL protein G sepharose beads for 1 h rotating at 4 °C.

Techniques: Binding Assay, Control

Journal: Nature Communications

Article Title: Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

doi: 10.1038/s41467-019-13616-1

Figure Lengend Snippet: a Top and side views of TNF (green ribbons) with UCB- 6876 bound (orange sticks). b Detail showing the electron density of UCB- 6876 and MPD bound within the TNF homotrimer. Monomers B (light green) and C (green) are shown surface rendered. Contour level of the electron density is set at 1 sigma. MPD occupies a space next to UCB- 6876 . Subsequent molecules described in this paper were modified to have chemical groups occupying the space where MPD was bound. c Detail of the compound-binding pocket within the TNF homotrimer, with key residues involved in binding highlighted (sticks and labels). d Side view of apo-TNF (left image) and UCB- 6876 bound TNF (right image) revealing the distorted AC receptor-binding site (selected residues involved in TNFR1 binding are highlighted red).

Article Snippet: TNFR1 immunoprecipitation was performed by incubating supernatant with 5 μg anti-TNFR1 antibody (R&D systems, #AB-225-PB) overnight while rotating at 4 °C, followed by the addition of 20 μL protein G sepharose beads for 1 h rotating at 4 °C.

Techniques: Modification, Binding Assay

Journal: Nature Communications

Article Title: Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

doi: 10.1038/s41467-019-13616-1

Figure Lengend Snippet: a Western blots measuring RIP1 ubiquitination following TNFR1 immunoprecipitation from Jurkat cells treated with TNF ± UCB- 9260 or etanercept. pNFkB expression was assessed, along with GAPDH for loading control. An isotype control antibody was included as an immunoprecipitation control (lane 5). b TNF (10 pM) or anti-TNFR1 agonist antibody (0.3 mg/mL)-driven NFkB activity was measured following UCB- 9260 treatment in Hek-293 cells using a reporter gene assay system. Representative concentration-response curves are shown. Percentage inhibitions for compound dilutions were calculated between a DMSO control and maximum inhibition (by excess biologic anti-TNF, or NFkB inhibitor–TPCA-1). Untransformed data shown in Supplementary Fig. . c UCB- 9260 inhibition of TNF (human or mouse) dependent cytotoxicity was measured in mouse L929 cells. Representative concentration-response curves are shown. Percentage inhibition was calculated between unstimulated wells (maximum signal), and wells with TNF, DMSO and actinomycin D (minimum signal). Untransformed data shown in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: TNFR1 immunoprecipitation was performed by incubating supernatant with 5 μg anti-TNFR1 antibody (R&D systems, #AB-225-PB) overnight while rotating at 4 °C, followed by the addition of 20 μL protein G sepharose beads for 1 h rotating at 4 °C.

Techniques: Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Expressing, Control, Activity Assay, Reporter Gene Assay, Concentration Assay, Inhibition