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mouse cytokine array kit panel a  (R&D Systems)
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R&D Systems mouse cytokine array kit panel a
Mouse Cytokine Array Kit Panel A, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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proteome profiler array  (R&D Systems)
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Proteome Profiler Array, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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panel a r d systems cat  (R&D Systems)
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Panel A R D Systems Cat, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human high sensitivity cytokine a premixed magnetic luminex® performance assay  (Bio-Techne corporation)
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Human High Sensitivity Cytokine A Premixed Magnetic Luminex® Performance Assay, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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proteome profiler mouse angiogenesis array kit  (Bio-Techne corporation)
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Proteome Profiler Mouse Angiogenesis Array Kit, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant hirudin  (Aniara Inc)
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Aniara Inc recombinant hirudin
Recombinant Hirudin, supplied by Aniara Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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biorad protein assay bio rad  (Bio-Rad)
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Biorad Protein Assay Bio Rad, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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uranyl acetate  (SERVA Electrophoresis)
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corticosterone eia kit  (Arbor Assays)
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paraformaldehyde (pfa)  (SERVA Electrophoresis)
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cell lines 293t cells  (DSMZ)
96
DSMZ cell lines 293t cells
Figure 1. EBOV-GP Is Released from GP-Expressing Cells in a Vesicle-Associated Manner (A) <t>293T</t> cells were transfected to express Ebola virus-glycoprotein (EBOV- GP). 24 hours post-transfection (hpt), supernatants were harvested and puri- fied with a protocol that pellets vesicles and virus-like particles (see STAR Methods). GP1 and GP2 in the purified media and cell lysates were analyzed using western blot. (B) 293T cells were transfected with EBOV-GP. 24 hours post-transfection, a linear sucrose gradient was established and overlaid with the cell culture supernatant of the GP-transfected cells. The supernatant was either left un- treated (top) or treated with 1% Triton X-100 (bottom). The supernatants were subsequently centrifuged at 50,000 3 g for 2 h at 4C. Afterward, ten fractions were collected from the bottom (fraction 10) to the top (fraction 1) of the gradient. The GP levels within the ten fractions were then analyzed using western blot using a rabbit serum that targets the GP1 subunit. (C) Supernatants of 293T cells were treated as in (A), but two sequential centrifugation steps were done to collect vesicles that pellet at 21,000 3 g or 100,000 3 g. Purified media and cells analyzed using western blot for exo- somal (CD81, TSG101) and cellular (CNX, actin) markers as well as GP. (D) Densitometric quantification of CD81 levels from four independent exper- iments. For each experiment, values for CD81 levels in cells and supernatants
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Image Search Results


Figure 1. EBOV-GP Is Released from GP-Expressing Cells in a Vesicle-Associated Manner (A) 293T cells were transfected to express Ebola virus-glycoprotein (EBOV- GP). 24 hours post-transfection (hpt), supernatants were harvested and puri- fied with a protocol that pellets vesicles and virus-like particles (see STAR Methods). GP1 and GP2 in the purified media and cell lysates were analyzed using western blot. (B) 293T cells were transfected with EBOV-GP. 24 hours post-transfection, a linear sucrose gradient was established and overlaid with the cell culture supernatant of the GP-transfected cells. The supernatant was either left un- treated (top) or treated with 1% Triton X-100 (bottom). The supernatants were subsequently centrifuged at 50,000 3 g for 2 h at 4C. Afterward, ten fractions were collected from the bottom (fraction 10) to the top (fraction 1) of the gradient. The GP levels within the ten fractions were then analyzed using western blot using a rabbit serum that targets the GP1 subunit. (C) Supernatants of 293T cells were treated as in (A), but two sequential centrifugation steps were done to collect vesicles that pellet at 21,000 3 g or 100,000 3 g. Purified media and cells analyzed using western blot for exo- somal (CD81, TSG101) and cellular (CNX, actin) markers as well as GP. (D) Densitometric quantification of CD81 levels from four independent exper- iments. For each experiment, values for CD81 levels in cells and supernatants

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 1. EBOV-GP Is Released from GP-Expressing Cells in a Vesicle-Associated Manner (A) 293T cells were transfected to express Ebola virus-glycoprotein (EBOV- GP). 24 hours post-transfection (hpt), supernatants were harvested and puri- fied with a protocol that pellets vesicles and virus-like particles (see STAR Methods). GP1 and GP2 in the purified media and cell lysates were analyzed using western blot. (B) 293T cells were transfected with EBOV-GP. 24 hours post-transfection, a linear sucrose gradient was established and overlaid with the cell culture supernatant of the GP-transfected cells. The supernatant was either left un- treated (top) or treated with 1% Triton X-100 (bottom). The supernatants were subsequently centrifuged at 50,000 3 g for 2 h at 4C. Afterward, ten fractions were collected from the bottom (fraction 10) to the top (fraction 1) of the gradient. The GP levels within the ten fractions were then analyzed using western blot using a rabbit serum that targets the GP1 subunit. (C) Supernatants of 293T cells were treated as in (A), but two sequential centrifugation steps were done to collect vesicles that pellet at 21,000 3 g or 100,000 3 g. Purified media and cells analyzed using western blot for exo- somal (CD81, TSG101) and cellular (CNX, actin) markers as well as GP. (D) Densitometric quantification of CD81 levels from four independent exper- iments. For each experiment, values for CD81 levels in cells and supernatants

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Expressing, Transfection, Virus, Western Blot, Cell Culture, Centrifugation

Figure 3. Tetherin Suppresses the Release of EBOV-GP (A) 293T cells were transfected to express EBOV-GP or an empty control plasmid (mock) and increasing amounts of tetherin. 24 hours post-trans- fection, GP levels in the media and in the cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (B) HeLa cells were transfected to express EBOV-GP and tetherin or an empty control plasmid. 24 hours post-transfection, cell culture media as well as cell lysates were analyzed as described in (A). (C) HeLa cells were transfected with a tetherin-specific or a non-targeting siRNA as control. 24 hours later, the cells were additionally transfected to express GP or a non-coding control plasmid. After another 24 h, GP and tetherin steady-state levels in cell culture media and in cell lysates were analyzed using western blot. The results were verified in at least three addi- tional experiments.

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 3. Tetherin Suppresses the Release of EBOV-GP (A) 293T cells were transfected to express EBOV-GP or an empty control plasmid (mock) and increasing amounts of tetherin. 24 hours post-trans- fection, GP levels in the media and in the cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (B) HeLa cells were transfected to express EBOV-GP and tetherin or an empty control plasmid. 24 hours post-transfection, cell culture media as well as cell lysates were analyzed as described in (A). (C) HeLa cells were transfected with a tetherin-specific or a non-targeting siRNA as control. 24 hours later, the cells were additionally transfected to express GP or a non-coding control plasmid. After another 24 h, GP and tetherin steady-state levels in cell culture media and in cell lysates were analyzed using western blot. The results were verified in at least three addi- tional experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Cell Culture

Figure 4. Determinants in Tetherin Impor- tant for Suppression of EBOV-GP Release (A) 293T cells were transfected to express EBOV- GP or a control plasmid and wild-type tetherin or the indicated tetherin mutants. 24 hours post- transfection, GP and tetherin levels in cell culture media and cell lysates were determined using western blot. (B) Representative confocal fluorescence images of 293T cells that were transfected to express the indicated tetherin variants. Tetherin was detected with a specific antibody and counterstained with an Alexa 633-conjugated secondary antibody. Nuclei of cells were visualized by DAPI. Scale bar, 40 mm. (C) 293T cells were transfected to express EBOV- GP or a control plasmid as well as Flag-tagged human (hu.), hamster (ham.), or alligator (allig.) tetherin. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (D) Same setup as in (C), but 293T cells were transfected to express HA-tagged human tetherin, artificial (art.) tetherin, Epomops franqueti (Epo.) tetherin, or Hypsignathus monstrosus (Hyp.) teth- erin together with GP or a non-coding control plasmid. All results presented were verified in at least three additional independent experiments.

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 4. Determinants in Tetherin Impor- tant for Suppression of EBOV-GP Release (A) 293T cells were transfected to express EBOV- GP or a control plasmid and wild-type tetherin or the indicated tetherin mutants. 24 hours post- transfection, GP and tetherin levels in cell culture media and cell lysates were determined using western blot. (B) Representative confocal fluorescence images of 293T cells that were transfected to express the indicated tetherin variants. Tetherin was detected with a specific antibody and counterstained with an Alexa 633-conjugated secondary antibody. Nuclei of cells were visualized by DAPI. Scale bar, 40 mm. (C) 293T cells were transfected to express EBOV- GP or a control plasmid as well as Flag-tagged human (hu.), hamster (ham.), or alligator (allig.) tetherin. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (D) Same setup as in (C), but 293T cells were transfected to express HA-tagged human tetherin, artificial (art.) tetherin, Epomops franqueti (Epo.) tetherin, or Hypsignathus monstrosus (Hyp.) teth- erin together with GP or a non-coding control plasmid. All results presented were verified in at least three additional independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot

Figure 5. The EBOV-GP RBD and TMD Are Determinants of GP Release (A) 293T cells were transfected to express EBOV-GP or the indicated GP mutants together with tetherin or a non-coding control plasmid. 24 hours post- transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in cell lysates were analyzed using western blot. (B) HeLa cells were transfected to express EBOV-GP or the indicated GP mutants together with a tetherin-specific siRNA or a non-targeting siRNA as negative control. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in cell lysates were analyzed using western blot. (C) HeLa cells were transfected to express EBOV-GP or the ELE variant together with GFP from a bicistronic plasmid or the respective control plasmid only expressing GFP (mock). 24 hours post-transfection, a proximity ligation assay was performed with antibodies against EBOV-GP1 and tetherin, respectively.

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 5. The EBOV-GP RBD and TMD Are Determinants of GP Release (A) 293T cells were transfected to express EBOV-GP or the indicated GP mutants together with tetherin or a non-coding control plasmid. 24 hours post- transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in cell lysates were analyzed using western blot. (B) HeLa cells were transfected to express EBOV-GP or the indicated GP mutants together with a tetherin-specific siRNA or a non-targeting siRNA as negative control. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in cell lysates were analyzed using western blot. (C) HeLa cells were transfected to express EBOV-GP or the ELE variant together with GFP from a bicistronic plasmid or the respective control plasmid only expressing GFP (mock). 24 hours post-transfection, a proximity ligation assay was performed with antibodies against EBOV-GP1 and tetherin, respectively.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot, Negative Control, Variant Assay, Expressing, Proximity Ligation Assay

Figure 6. Ebolavirus GP Release Is Associ- ated with Increased Viral Pathogenicity (A) 293T cells were transfected to express EBOV- GP, SUDV-GP, TAFV-GP, or RESTV-GP together with tetherin or a non-coding control plasmid. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (B) HeLa cells were transfected to express EBOV- GP, SUDV-GP, TAFV-GP, or RESTV-GP, and GP levels in cell culture media and cell lysates were quantified using western blot. (C) 293T cells were transfected to express EBOV- GP or the GP-A82V variant derived from the 2014 Ebola outbreak in West Africa together with teth- erin or a non-coding control plasmid. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (D) HeLa cells were transfected to express EBOV- GP or the GP-A82V variant and GP levels in cell culture media and cell lysates were quantified us- ing western blot. All results were verified in at least three independent experiments.

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 6. Ebolavirus GP Release Is Associ- ated with Increased Viral Pathogenicity (A) 293T cells were transfected to express EBOV- GP, SUDV-GP, TAFV-GP, or RESTV-GP together with tetherin or a non-coding control plasmid. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (B) HeLa cells were transfected to express EBOV- GP, SUDV-GP, TAFV-GP, or RESTV-GP, and GP levels in cell culture media and cell lysates were quantified using western blot. (C) 293T cells were transfected to express EBOV- GP or the GP-A82V variant derived from the 2014 Ebola outbreak in West Africa together with teth- erin or a non-coding control plasmid. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (D) HeLa cells were transfected to express EBOV- GP or the GP-A82V variant and GP levels in cell culture media and cell lysates were quantified us- ing western blot. All results were verified in at least three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot, Variant Assay, Derivative Assay

Figure 7. GP-Virosomes Are Decoys for EBOV-Neutralizing Anti- bodies and Have Immunomodulatory Functions (A) 293T cells were transduced with MLV-pseudotypes bearing EBOV-GP or VSVG, mixed with the indicated cell culture supernatants and the indicated amounts of EBOV-GP-neutralizing antibody (KZ52). The transduction effi- ciency of MLV pseudotypes mixed with the indicated supernatants in the absence of antibody was set to 100%. Shown is the mean relative transduction efficiency (SEM, n = 5, triplicate transductions). We used a two-way ANOVA with Dunnett’s multiple-comparison test to determine statistical significance (*p < 0.05 and ***p < 0.001). (B) Similar setup as in (A), but 293T cells were transduced with transcription and replication competent EBOV-like particles (trVLP) and mixed with the indicated cell culture supernatants and increasing amounts of the KZ52 anti- body. The transduction efficiency of trVLPs mixed with the indicated super- natants in the absence of antibody was set to 100%. Shown is the mean relative transduction efficiency (SEM, n = 3, triplicate transductions). We used a two-way ANOVA with Dunnett’s multiple-comparison test to determine statistical significance (*p < 0.05 and **p < 0.01). (C) Primary human monocyte-derived macrophages were treated with the indicated cell culture supernatants. 24 hours later, relative cytokine levels were quantified with a cytokine array. The floating bar plot shows the mean as well as minimum and maximum levels for each cytokine measured from experi- ments with three independent macrophage donors. We used a two-way ANOVA with Fisher’s least significant difference (LSD) test to determine sta- tistical significance (*p < 0.05 and **p < 0.01). Right: representative primary array spots of the eight cytokines analyzed.

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 7. GP-Virosomes Are Decoys for EBOV-Neutralizing Anti- bodies and Have Immunomodulatory Functions (A) 293T cells were transduced with MLV-pseudotypes bearing EBOV-GP or VSVG, mixed with the indicated cell culture supernatants and the indicated amounts of EBOV-GP-neutralizing antibody (KZ52). The transduction effi- ciency of MLV pseudotypes mixed with the indicated supernatants in the absence of antibody was set to 100%. Shown is the mean relative transduction efficiency (SEM, n = 5, triplicate transductions). We used a two-way ANOVA with Dunnett’s multiple-comparison test to determine statistical significance (*p < 0.05 and ***p < 0.001). (B) Similar setup as in (A), but 293T cells were transduced with transcription and replication competent EBOV-like particles (trVLP) and mixed with the indicated cell culture supernatants and increasing amounts of the KZ52 anti- body. The transduction efficiency of trVLPs mixed with the indicated super- natants in the absence of antibody was set to 100%. Shown is the mean relative transduction efficiency (SEM, n = 3, triplicate transductions). We used a two-way ANOVA with Dunnett’s multiple-comparison test to determine statistical significance (*p < 0.05 and **p < 0.01). (C) Primary human monocyte-derived macrophages were treated with the indicated cell culture supernatants. 24 hours later, relative cytokine levels were quantified with a cytokine array. The floating bar plot shows the mean as well as minimum and maximum levels for each cytokine measured from experi- ments with three independent macrophage donors. We used a two-way ANOVA with Fisher’s least significant difference (LSD) test to determine sta- tistical significance (*p < 0.05 and **p < 0.01). Right: representative primary array spots of the eight cytokines analyzed.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transduction, Cell Culture, Comparison, Derivative Assay