Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Humanized hu/mTNFR1-k/i mouse models. Schematic representation of the homologous recombination in the TNFR1 gene locus. Shown is the wild-type genomic locus (A), the targeted locus, including the neo cassette (B), and the genomic locus of chimeric mice (Cre’d locus; C).

Article Snippet: Then, 0.5 × 10 6 cells were incubated with antibodies against mouse TNFR1 (HP8002; Hycult Biotec), human TNFR1 (HP9002; Hycult Biotec), mouse TNFR2 (HP8003; Hycult Biotec), and human TNFR2 (HP9003; Hycult Biotec) for 45 min on ice.

Techniques: Homologous Recombination

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Expression of transgenic hu/mTNFR1-k/i and hu/mTNFR2-k/i in homozygous mice. Splenocytes or thymocytes were isolated from C57BL/6J wild-type (A and B), hu/mTNFR1-k/i (A), or hu/mTNFR2-k/i (B) mice. Then mouse TNFR1 (HP8002) and human TNFR1 (HP9002) (A) or mouse TNFR2 (HP8003) and human TNFR2 (HP9003) (B) expression was analyzed by flow cytometry using subgates for CD45R+ B cells, CD68+ macrophages (splenocytes), and CD8+ thymocytes. Gray histograms show the isotype controls; black lines indicate signals measured for TNFR1 or TNFR2 expression. Data are presented as normalized to unit area.

Article Snippet: Then, 0.5 × 10 6 cells were incubated with antibodies against mouse TNFR1 (HP8002; Hycult Biotec), human TNFR1 (HP9002; Hycult Biotec), mouse TNFR2 (HP8003; Hycult Biotec), and human TNFR2 (HP9003; Hycult Biotec) for 45 min on ice.

Techniques: Expressing, Transgenic Assay, Isolation, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Expression of transgenic hu/mTNFR1-k/i and hu/mTNFR2-k/i in primary cells isolated from homozygous mice. Primary MEFs were isolated from wild-type (A and B), hu/mTNFR1-k/i (A), or hu/mTNFR2-k/i (B) C57BL/6 mice. Expression of mouse TNFR1 (HP8002) and human TNFR1 (HP9002) (A) or mouse TNFR2 (HP8003) and human TNFR2 (HP9003) (B) was analyzed by flow cytometry. Data are presented as normalized to unit area. (C and D) Brain tissue isolated from wild-type (C and D), hu/mTNFR1-k/I (C), or hu/mTNFR2-k/i (D) C57BL/6 mice. Tissue was lyzed and expression of huTNFR1 (wild-type, hu/mTNFR1-k/i, H398) or huTNFR2 (wild-type, hu/mTNFR2-k/i, MR2-1) was analyzed by Western blot.

Article Snippet: Then, 0.5 × 10 6 cells were incubated with antibodies against mouse TNFR1 (HP8002; Hycult Biotec), human TNFR1 (HP9002; Hycult Biotec), mouse TNFR2 (HP8003; Hycult Biotec), and human TNFR2 (HP9003; Hycult Biotec) for 45 min on ice.

Techniques: Expressing, Transgenic Assay, Isolation, Flow Cytometry, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Mechanism of TNFR1 antagonist ATROSAB and TNFR2 agonist EHD2–scTNFR2-mediated neuroprotection. (A) TNFR1 signaling induces neurodegeneration and inflammation, whereas TNFR2 signaling promotes neuroprotection. (B) ATROSAB, an antagonistic TNFR1-selective antibody, can block TNFR1 signaling, leading to prevention of neurodegeneration and inflammation. More importantly, in the presence of ATROSAB, tmTNF stays free to activate neuroprotective signaling via TNFR2. Our data indicate that, by blocking TNFR1, ATROSAB shifts the endogenous tmTNF signaling toward neuroprotective TNFR2 signaling. Similarly, the TNFR2 agonist EHD2–scTNFR2 can directly activate TNFR2 and thereby induce neuroprotection.

Article Snippet: Then, 0.5 × 10 6 cells were incubated with antibodies against mouse TNFR1 (HP8002; Hycult Biotec), human TNFR1 (HP9002; Hycult Biotec), mouse TNFR2 (HP8003; Hycult Biotec), and human TNFR2 (HP9003; Hycult Biotec) for 45 min on ice.

Techniques: Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Humanized hu/mTNFR1-k/i mouse models. Schematic representation of the homologous recombination in the TNFR1 gene locus. Shown is the wild-type genomic locus (A), the targeted locus, including the neo cassette (B), and the genomic locus of chimeric mice (Cre’d locus; C).

Article Snippet: After transfer to PVDF membranes (Millipore), membranes were blocked for 1 h with 1% I-blocker (Tropix) in TBS containing 0.0625% Tween 20 and subsequently incubated overnight with primary antibody at 4 °C [anti-human TNFR1 was purchased from Hycult Biotech (H398); anti-human TNFR2 was from Abcam (MR2-1)].

Techniques: Homologous Recombination

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Expression of transgenic hu/mTNFR1-k/i and hu/mTNFR2-k/i in homozygous mice. Splenocytes or thymocytes were isolated from C57BL/6J wild-type (A and B), hu/mTNFR1-k/i (A), or hu/mTNFR2-k/i (B) mice. Then mouse TNFR1 (HP8002) and human TNFR1 (HP9002) (A) or mouse TNFR2 (HP8003) and human TNFR2 (HP9003) (B) expression was analyzed by flow cytometry using subgates for CD45R+ B cells, CD68+ macrophages (splenocytes), and CD8+ thymocytes. Gray histograms show the isotype controls; black lines indicate signals measured for TNFR1 or TNFR2 expression. Data are presented as normalized to unit area.

Article Snippet: After transfer to PVDF membranes (Millipore), membranes were blocked for 1 h with 1% I-blocker (Tropix) in TBS containing 0.0625% Tween 20 and subsequently incubated overnight with primary antibody at 4 °C [anti-human TNFR1 was purchased from Hycult Biotech (H398); anti-human TNFR2 was from Abcam (MR2-1)].

Techniques: Expressing, Transgenic Assay, Isolation, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Expression of transgenic hu/mTNFR1-k/i and hu/mTNFR2-k/i in primary cells isolated from homozygous mice. Primary MEFs were isolated from wild-type (A and B), hu/mTNFR1-k/i (A), or hu/mTNFR2-k/i (B) C57BL/6 mice. Expression of mouse TNFR1 (HP8002) and human TNFR1 (HP9002) (A) or mouse TNFR2 (HP8003) and human TNFR2 (HP9003) (B) was analyzed by flow cytometry. Data are presented as normalized to unit area. (C and D) Brain tissue isolated from wild-type (C and D), hu/mTNFR1-k/I (C), or hu/mTNFR2-k/i (D) C57BL/6 mice. Tissue was lyzed and expression of huTNFR1 (wild-type, hu/mTNFR1-k/i, H398) or huTNFR2 (wild-type, hu/mTNFR2-k/i, MR2-1) was analyzed by Western blot.

Article Snippet: After transfer to PVDF membranes (Millipore), membranes were blocked for 1 h with 1% I-blocker (Tropix) in TBS containing 0.0625% Tween 20 and subsequently incubated overnight with primary antibody at 4 °C [anti-human TNFR1 was purchased from Hycult Biotech (H398); anti-human TNFR2 was from Abcam (MR2-1)].

Techniques: Expressing, Transgenic Assay, Isolation, Flow Cytometry, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration

doi: 10.1073/pnas.1605195113

Figure Lengend Snippet: Mechanism of TNFR1 antagonist ATROSAB and TNFR2 agonist EHD2–scTNFR2-mediated neuroprotection. (A) TNFR1 signaling induces neurodegeneration and inflammation, whereas TNFR2 signaling promotes neuroprotection. (B) ATROSAB, an antagonistic TNFR1-selective antibody, can block TNFR1 signaling, leading to prevention of neurodegeneration and inflammation. More importantly, in the presence of ATROSAB, tmTNF stays free to activate neuroprotective signaling via TNFR2. Our data indicate that, by blocking TNFR1, ATROSAB shifts the endogenous tmTNF signaling toward neuroprotective TNFR2 signaling. Similarly, the TNFR2 agonist EHD2–scTNFR2 can directly activate TNFR2 and thereby induce neuroprotection.

Article Snippet: After transfer to PVDF membranes (Millipore), membranes were blocked for 1 h with 1% I-blocker (Tropix) in TBS containing 0.0625% Tween 20 and subsequently incubated overnight with primary antibody at 4 °C [anti-human TNFR1 was purchased from Hycult Biotech (H398); anti-human TNFR2 was from Abcam (MR2-1)].

Techniques: Blocking Assay

Journal: The American Journal of Pathology

Article Title: The Critical Role of TAK1 in Accentuated Epithelial to Mesenchymal Transition in Obliterative Bronchiolitis after Lung Transplantation

doi: 10.1016/j.ajpath.2012.02.022

Figure Lengend Snippet: TNF-α accentuates TGF-β1–driven EMT via TNFR1. A: Flow cytometry analysis demonstrates that PBECs express TNFR1 on their cell surface, but express little to no TNFR2. B: Stimulation of PBECs (n = 6) with soluble TNF-α (TNFsol), membrane-bound TNF-α (TNFcys), TNFR1-specific mutant (Cys-TNF32W/86T) membrane-bound TNF (TNFcysR1), or TNFR2-specific mutant (Cys-TNF143N/145R) membrane-bound TNF (TNFcysR2) (all 20 ng/mL) for 72 hours alone has no effect on the expression of E-cadherin, vimentin, or fibronectin. Stimulation, however, with TNFsol, TNFcys, or TNFcysR1, but not TNFcysR2, increases pro-MMP-9 secretion. TGF-β1 (10 ng/mL) down-regulates the expression of E-cadherin, increases the expression of vimentin and fibronectin, and increases the secretion of pro-MMP-9. Co-stimulation of the cells with TGF-β1 and TNFsol, TNFcys, or TNFcysR1, but not TNFcysR2, accentuates the changes in EMT marker expression compared to TGF-β1 alone. C: Co-stimulation of PBECs with TGF-β1 and TNFsol, TNFcys, or TNFcysR1, but not TNFcysR2, accentuates changes in cell morphology and EMT marker expression compared to TGF-β1 alone. FL1-H, height of fluorescence intensity; FSC-H, height of forward scatter; SSC-H, height of side scatter.

Article Snippet: TNFR1 (HM2020; Hycult, Uden, The Netherlands) and TNFR2 (HM2007; Hycult) 21 primary antibodies were used to detect receptor expression by flow cytometry.

Techniques: Flow Cytometry, Mutagenesis, Expressing, Marker, Fluorescence