Blocking Assay:Article Title: Repurpose of Ritlecitinib Reduces Neointima Formation and Enhances Endothelial Recovery Following Vascular Injury
Article Snippet: .. In separate experimental groups, additional interventions were applied: bevacizumab (5 mg/kg, i.v, twice weekly; Avastin, Genentech) was used to block VEGF-A signaling, while recombinant human thrombospondin-1 (TSP-1, 5 μg/graft; R&D Systems, #3074-TH) was delivered by i.v injection on days 0 and 7 to enhance TSP-1 activity. ..
Recombinant:Article Title: Repurpose of Ritlecitinib Reduces Neointima Formation and Enhances Endothelial Recovery Following Vascular Injury
Article Snippet: .. In separate experimental groups, additional interventions were applied: bevacizumab (5 mg/kg, i.v, twice weekly; Avastin, Genentech) was used to block VEGF-A signaling, while recombinant human thrombospondin-1 (TSP-1, 5 μg/graft; R&D Systems, #3074-TH) was delivered by i.v injection on days 0 and 7 to enhance TSP-1 activity. ..
Article Title: Effects of TAZ on human dental pulp stem cell proliferation and migration.
Article Snippet: The invaded cells on the lower membrane were fixed for 10 min with 4% paraformaldehyde at 4 ̊C, stained at 37 ̊C for 10 min with 5 μg/ml Hoechst 33342 and counted under an upright fluorescence microscope (Olympus Corporation). .. Treatment with recombinant human thrombospondin 1 (r‐hTHBS1). hDPSCs were seeded into 6-well plates at a density of 2x105 cells/well and cultured for 24 h. Subsequently, cells were incubated at 37 ̊C for 10 min with r‐hTHBS1, a TGF-β activator (200 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA). ..
Article Title: Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.
Article Snippet: .. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [29–31]. .. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Article Title: Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells
Article Snippet: .. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [ – ]. .. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Article Title: The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-HTRA1 Abcam Cat#ab38611; RRID: AB_733053 Goat polyclonal anti-IBA1 Abcam Cat#ab5076; RRID: AB_2224402 Goat polyclonal anti-TSP1 Abcam Cat#ab111230; RRID: AB_10867429 Monoclonal anti-FLAG M2 antibody Sigma-Aldrich Cat#F3165; RRID: AB_259529 Duolink In Situ PLA Probe anti-Rabbit PLUS antibody Sigma-Aldrich Cat#DUO92002; RRID: AB_2810940 Duolink In Situ PLA Probe anti-Mouse MINUS Antibody Sigma-Aldrich Cat#DUO92004; RRID: AB_2713942 Mouse Monoclonal anti-V5 Tag Thermo Fisher Scientific Cat#R960-25; RRID: AB_2556564 Rabbit polyclonal anti-Myc tag Abcam Cat#ab9106; RRID: AB_307014 Mouse monoclonal anti-CD36 Abcam Cat#ab17044; RRID: AB_443600 Mouse monoclonal anti-CD47 Abcam Cat#ab3283; RRID: AB_303671 Mouse monoclonal anti-TSP1 Thermo Fisher Scientific Cat#MA5-13390; RRID: AB_10979326 Mouse IgG1 Isotype Control R&D Systems Cat#MAB002; RRID: AB_357344 Goat IgG1 Isotype Control R&D Systems Cat#AB-108-C; RRID: AB_354267 Rabbit polyclonal anti-PU.1 Thermo Fisher Scientific Cat#PA5-17505; RRID: AB_10989141 Goat polyclonal anti-Otx2 R&D Systems Cat#AF1979; RRID: AB_2157172 Goat polyclonal anti-mouse Osteopontin R&D Systems Cat#AF808; RRID: AB_2194992 Goat polyclonal anti-human Osteopontin R&D Systems Cat#AF1433; RRID: AB_354791 Rabbit polyclonal anti-Iba1 Wako Cat#019-19741; RRID: AB_839504 Rat monoclonal anti-CD102 BD Biosciences Cat#553325; RRID: AB_394783 Biological Samples Peripheral blood from human donors CHNO des Quinze- Vingts, Paris, France N/A Post-mortem RPE/choroid/sclera and retinal pieces from healthy and AMD donors Minnesota Lions Eye Bank, USA N/A Healthy porcine eyes Guy Harang slaughterhouse, Houdan, France N/A Chemicals, Peptides, and Recombinant Proteins Recombinant Human Thrombospondin-1 Protein R&D Systems Cat3074-TH-050 Recombinant Human Thrombospondin-1 C-terminal domain (Cter) with a FLAG-epitope at the N terminus GTP Technology, Labege, France N/A Recombinant Human HTRA1 Protein R&D Systems Cat#2916-SE-020 Control peptide (KRFYGGMWKK) Genepep, France N/A 4N1K CD47-activating peptide (KRFYVVMWKK) Genepep, France N/A Control bi- peptide Ac- KVRMKF YVWKGGGGGGGGKVRMKF YVWK -NH2 Genepep, France N/A Bi4N1K peptide (with PEG spacer) Ac-KRFYVVMWKK-PEG7-KRFYVVMWKK-NH2 PEG7 = NH2-(CH2CH2O)7-CH2CH2COOH Genepep, France N/A Bi4N1K peptide (with 8G spacer) Ac-KRFYVVMWKKGGGGG GGGKRFYVVMWKK-NH2 Genepep, France N/A (Continued on next page) e1 Immunity 53, 429–441.e1–e8, August 18, 2020 .. REAGENT or RESOURCE SOURCE IDENTIFIER HTRA1 inhibitor DPMFKLboroV Roche Innovation Center, Basel, Swiss www.roche.com Critical Commercial Assays EasySep Mouse Monocyte Isolation Kit STEMCELL Technologies Cat#19861 EasySep Human Monocyte Enrichment Kit STEMCELL Technologies Cat#19059 Human HTRA1 Elisa Elabscience Cat#E-EL-H0423 Humain OPN Elisa R&D Systems Cat#DOST00 MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panels Millipore Cat#HCYTOMAG Experimental Models: Cell Lines COS-7 cell line ATCC Cat#CRL-1651, RRID: CVCL_0224 Experimental Models: Organisms/Strains Mouse: C57BL/6J (rd8 mutation free) The Jackson Laboratory RRID: IMSR_JAX:000664 Mouse: Opn / (rd8 mutation free) The Jackson Laboratory B6.129S6(Cg)-Spp1tm1Blh/J Stock No: 004936 Mouse: Thbs1 / (rd8 mutation free) The Jackson Laboratory B6.129S2-Thbs1tm1Hyn/J Stock No: 006141 Oligonucleotides Taqman primers for HTRA1 Thermo Fisher Scientific Cat#Hs01016151_m1 Taqman primers for ARMS2 Thermo Fisher Scientific Cat#Hs01394203_m1 Primers for human ARMS2 ARMS2 forward 5¢-TCGGTGGTT CCTGTGTCCTTCATT-3¢, ARMS2 reverse 5¢-TCACCTTGCTGCAG TGTGGATGA-3¢ Integrated DNA Technologies, Inc N/A Primers for human OPNSPP1 forward 5¢-GCACAGGTGATGC CTAGGAG-3¢, SPP1 reverse 5¢- ACCAGACTCGTCTCAGGAG-3¢ Integrated DNA Technologies, Inc N/A Primers for human PLEKHA1 PLEKHA1 forward 5¢-GCCCTGAA GAGATGCACAGT-3¢, PLEKHA1 reverse 5¢-AAGGGTTCGACAGCCTTCTG-3¢ Integrated DNA Technologies, Inc N/A Primers for Mus musculus Opn Spp1 forward 5¢-TCTTCTGCGG CAGGCATT-3¢Spp1 reverse 5¢-TGC CAATCTCATGGTCGT-3¢ Integrated DNA Technologies, Inc N/A Recombinant DNA Plasmid: pCX_CD47-V5; human CD47 with a C-terminal V5 flag This paper Genecust, Boynes, France N/A Plasmid: pCX_CD47-Myc; human CD47 with a C-terminal Myc flag This paper Genecust, Boynes, France N/A Software and Algorithms ProteinScape 2.1 Bruker Daltonics www.bruker.com GraphPad Prism GraphPad Software www.graphpad.com/ Compute pI/Mw Expasy https://web.expasy.org/compute_pi/ Data Analysis 3.4 Bruker www.bruker.com Mascot Server 2.2.07 Matrix Science www.matrixscience.com/ PEAKS Studio 8.5 Bioinformatics Solutions Inc. www.bioinfor.com MetaMorph Molecular Devices www.moleculardevices.com HCS iDev Cell Analysis Software Thermo Fisher Scientific Cat#SX000041A (Continued on next page) Immunity 53, 429–441.e1–e8, August 18, 2020 e2
Injection:Article Title: Repurpose of Ritlecitinib Reduces Neointima Formation and Enhances Endothelial Recovery Following Vascular Injury
Article Snippet: .. In separate experimental groups, additional interventions were applied: bevacizumab (5 mg/kg, i.v, twice weekly; Avastin, Genentech) was used to block VEGF-A signaling, while recombinant human thrombospondin-1 (TSP-1, 5 μg/graft; R&D Systems, #3074-TH) was delivered by i.v injection on days 0 and 7 to enhance TSP-1 activity. ..
Activity Assay:Article Title: Repurpose of Ritlecitinib Reduces Neointima Formation and Enhances Endothelial Recovery Following Vascular Injury
Article Snippet: .. In separate experimental groups, additional interventions were applied: bevacizumab (5 mg/kg, i.v, twice weekly; Avastin, Genentech) was used to block VEGF-A signaling, while recombinant human thrombospondin-1 (TSP-1, 5 μg/graft; R&D Systems, #3074-TH) was delivered by i.v injection on days 0 and 7 to enhance TSP-1 activity. ..
Cell Culture:Article Title: Effects of TAZ on human dental pulp stem cell proliferation and migration.
Article Snippet: The invaded cells on the lower membrane were fixed for 10 min with 4% paraformaldehyde at 4 ̊C, stained at 37 ̊C for 10 min with 5 μg/ml Hoechst 33342 and counted under an upright fluorescence microscope (Olympus Corporation). .. Treatment with recombinant human thrombospondin 1 (r‐hTHBS1). hDPSCs were seeded into 6-well plates at a density of 2x105 cells/well and cultured for 24 h. Subsequently, cells were incubated at 37 ̊C for 10 min with r‐hTHBS1, a TGF-β activator (200 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA). ..
Incubation:Article Title: Effects of TAZ on human dental pulp stem cell proliferation and migration.
Article Snippet: The invaded cells on the lower membrane were fixed for 10 min with 4% paraformaldehyde at 4 ̊C, stained at 37 ̊C for 10 min with 5 μg/ml Hoechst 33342 and counted under an upright fluorescence microscope (Olympus Corporation). .. Treatment with recombinant human thrombospondin 1 (r‐hTHBS1). hDPSCs were seeded into 6-well plates at a density of 2x105 cells/well and cultured for 24 h. Subsequently, cells were incubated at 37 ̊C for 10 min with r‐hTHBS1, a TGF-β activator (200 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA). ..
Article Title: Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.
Article Snippet: .. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [29–31]. .. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Article Title: Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells
Article Snippet: .. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [ – ]. .. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Purification:Article Title: Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.
Article Snippet: .. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [29–31]. .. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Article Title: Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells
Article Snippet: .. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [ – ]. .. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Enzyme-linked Immunosorbent Assay:
In Situ:Article Title: The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-HTRA1 Abcam Cat#ab38611; RRID: AB_733053 Goat polyclonal anti-IBA1 Abcam Cat#ab5076; RRID: AB_2224402 Goat polyclonal anti-TSP1 Abcam Cat#ab111230; RRID: AB_10867429 Monoclonal anti-FLAG M2 antibody Sigma-Aldrich Cat#F3165; RRID: AB_259529 Duolink In Situ PLA Probe anti-Rabbit PLUS antibody Sigma-Aldrich Cat#DUO92002; RRID: AB_2810940 Duolink In Situ PLA Probe anti-Mouse MINUS Antibody Sigma-Aldrich Cat#DUO92004; RRID: AB_2713942 Mouse Monoclonal anti-V5 Tag Thermo Fisher Scientific Cat#R960-25; RRID: AB_2556564 Rabbit polyclonal anti-Myc tag Abcam Cat#ab9106; RRID: AB_307014 Mouse monoclonal anti-CD36 Abcam Cat#ab17044; RRID: AB_443600 Mouse monoclonal anti-CD47 Abcam Cat#ab3283; RRID: AB_303671 Mouse monoclonal anti-TSP1 Thermo Fisher Scientific Cat#MA5-13390; RRID: AB_10979326 Mouse IgG1 Isotype Control R&D Systems Cat#MAB002; RRID: AB_357344 Goat IgG1 Isotype Control R&D Systems Cat#AB-108-C; RRID: AB_354267 Rabbit polyclonal anti-PU.1 Thermo Fisher Scientific Cat#PA5-17505; RRID: AB_10989141 Goat polyclonal anti-Otx2 R&D Systems Cat#AF1979; RRID: AB_2157172 Goat polyclonal anti-mouse Osteopontin R&D Systems Cat#AF808; RRID: AB_2194992 Goat polyclonal anti-human Osteopontin R&D Systems Cat#AF1433; RRID: AB_354791 Rabbit polyclonal anti-Iba1 Wako Cat#019-19741; RRID: AB_839504 Rat monoclonal anti-CD102 BD Biosciences Cat#553325; RRID: AB_394783 Biological Samples Peripheral blood from human donors CHNO des Quinze- Vingts, Paris, France N/A Post-mortem RPE/choroid/sclera and retinal pieces from healthy and AMD donors Minnesota Lions Eye Bank, USA N/A Healthy porcine eyes Guy Harang slaughterhouse, Houdan, France N/A Chemicals, Peptides, and Recombinant Proteins Recombinant Human Thrombospondin-1 Protein R&D Systems Cat3074-TH-050 Recombinant Human Thrombospondin-1 C-terminal domain (Cter) with a FLAG-epitope at the N terminus GTP Technology, Labege, France N/A Recombinant Human HTRA1 Protein R&D Systems Cat#2916-SE-020 Control peptide (KRFYGGMWKK) Genepep, France N/A 4N1K CD47-activating peptide (KRFYVVMWKK) Genepep, France N/A Control bi- peptide Ac- KVRMKF YVWKGGGGGGGGKVRMKF YVWK -NH2 Genepep, France N/A Bi4N1K peptide (with PEG spacer) Ac-KRFYVVMWKK-PEG7-KRFYVVMWKK-NH2 PEG7 = NH2-(CH2CH2O)7-CH2CH2COOH Genepep, France N/A Bi4N1K peptide (with 8G spacer) Ac-KRFYVVMWKKGGGGG GGGKRFYVVMWKK-NH2 Genepep, France N/A (Continued on next page) e1 Immunity 53, 429–441.e1–e8, August 18, 2020 .. REAGENT or RESOURCE SOURCE IDENTIFIER HTRA1 inhibitor DPMFKLboroV Roche Innovation Center, Basel, Swiss www.roche.com Critical Commercial Assays EasySep Mouse Monocyte Isolation Kit STEMCELL Technologies Cat#19861 EasySep Human Monocyte Enrichment Kit STEMCELL Technologies Cat#19059 Human HTRA1 Elisa Elabscience Cat#E-EL-H0423 Humain OPN Elisa R&D Systems Cat#DOST00 MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panels Millipore Cat#HCYTOMAG Experimental Models: Cell Lines COS-7 cell line ATCC Cat#CRL-1651, RRID: CVCL_0224 Experimental Models: Organisms/Strains Mouse: C57BL/6J (rd8 mutation free) The Jackson Laboratory RRID: IMSR_JAX:000664 Mouse: Opn / (rd8 mutation free) The Jackson Laboratory B6.129S6(Cg)-Spp1tm1Blh/J Stock No: 004936 Mouse: Thbs1 / (rd8 mutation free) The Jackson Laboratory B6.129S2-Thbs1tm1Hyn/J Stock No: 006141 Oligonucleotides Taqman primers for HTRA1 Thermo Fisher Scientific Cat#Hs01016151_m1 Taqman primers for ARMS2 Thermo Fisher Scientific Cat#Hs01394203_m1 Primers for human ARMS2 ARMS2 forward 5¢-TCGGTGGTT CCTGTGTCCTTCATT-3¢, ARMS2 reverse 5¢-TCACCTTGCTGCAG TGTGGATGA-3¢ Integrated DNA Technologies, Inc N/A Primers for human OPNSPP1 forward 5¢-GCACAGGTGATGC CTAGGAG-3¢, SPP1 reverse 5¢- ACCAGACTCGTCTCAGGAG-3¢ Integrated DNA Technologies, Inc N/A Primers for human PLEKHA1 PLEKHA1 forward 5¢-GCCCTGAA GAGATGCACAGT-3¢, PLEKHA1 reverse 5¢-AAGGGTTCGACAGCCTTCTG-3¢ Integrated DNA Technologies, Inc N/A Primers for Mus musculus Opn Spp1 forward 5¢-TCTTCTGCGG CAGGCATT-3¢Spp1 reverse 5¢-TGC CAATCTCATGGTCGT-3¢ Integrated DNA Technologies, Inc N/A Recombinant DNA Plasmid: pCX_CD47-V5; human CD47 with a C-terminal V5 flag This paper Genecust, Boynes, France N/A Plasmid: pCX_CD47-Myc; human CD47 with a C-terminal Myc flag This paper Genecust, Boynes, France N/A Software and Algorithms ProteinScape 2.1 Bruker Daltonics www.bruker.com GraphPad Prism GraphPad Software www.graphpad.com/ Compute pI/Mw Expasy https://web.expasy.org/compute_pi/ Data Analysis 3.4 Bruker www.bruker.com Mascot Server 2.2.07 Matrix Science www.matrixscience.com/ PEAKS Studio 8.5 Bioinformatics Solutions Inc. www.bioinfor.com MetaMorph Molecular Devices www.moleculardevices.com HCS iDev Cell Analysis Software Thermo Fisher Scientific Cat#SX000041A (Continued on next page) Immunity 53, 429–441.e1–e8, August 18, 2020 e2
Proximity Ligation Assay:Article Title: The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-HTRA1 Abcam Cat#ab38611; RRID: AB_733053 Goat polyclonal anti-IBA1 Abcam Cat#ab5076; RRID: AB_2224402 Goat polyclonal anti-TSP1 Abcam Cat#ab111230; RRID: AB_10867429 Monoclonal anti-FLAG M2 antibody Sigma-Aldrich Cat#F3165; RRID: AB_259529 Duolink In Situ PLA Probe anti-Rabbit PLUS antibody Sigma-Aldrich Cat#DUO92002; RRID: AB_2810940 Duolink In Situ PLA Probe anti-Mouse MINUS Antibody Sigma-Aldrich Cat#DUO92004; RRID: AB_2713942 Mouse Monoclonal anti-V5 Tag Thermo Fisher Scientific Cat#R960-25; RRID: AB_2556564 Rabbit polyclonal anti-Myc tag Abcam Cat#ab9106; RRID: AB_307014 Mouse monoclonal anti-CD36 Abcam Cat#ab17044; RRID: AB_443600 Mouse monoclonal anti-CD47 Abcam Cat#ab3283; RRID: AB_303671 Mouse monoclonal anti-TSP1 Thermo Fisher Scientific Cat#MA5-13390; RRID: AB_10979326 Mouse IgG1 Isotype Control R&D Systems Cat#MAB002; RRID: AB_357344 Goat IgG1 Isotype Control R&D Systems Cat#AB-108-C; RRID: AB_354267 Rabbit polyclonal anti-PU.1 Thermo Fisher Scientific Cat#PA5-17505; RRID: AB_10989141 Goat polyclonal anti-Otx2 R&D Systems Cat#AF1979; RRID: AB_2157172 Goat polyclonal anti-mouse Osteopontin R&D Systems Cat#AF808; RRID: AB_2194992 Goat polyclonal anti-human Osteopontin R&D Systems Cat#AF1433; RRID: AB_354791 Rabbit polyclonal anti-Iba1 Wako Cat#019-19741; RRID: AB_839504 Rat monoclonal anti-CD102 BD Biosciences Cat#553325; RRID: AB_394783 Biological Samples Peripheral blood from human donors CHNO des Quinze- Vingts, Paris, France N/A Post-mortem RPE/choroid/sclera and retinal pieces from healthy and AMD donors Minnesota Lions Eye Bank, USA N/A Healthy porcine eyes Guy Harang slaughterhouse, Houdan, France N/A Chemicals, Peptides, and Recombinant Proteins Recombinant Human Thrombospondin-1 Protein R&D Systems Cat3074-TH-050 Recombinant Human Thrombospondin-1 C-terminal domain (Cter) with a FLAG-epitope at the N terminus GTP Technology, Labege, France N/A Recombinant Human HTRA1 Protein R&D Systems Cat#2916-SE-020 Control peptide (KRFYGGMWKK) Genepep, France N/A 4N1K CD47-activating peptide (KRFYVVMWKK) Genepep, France N/A Control bi- peptide Ac- KVRMKF YVWKGGGGGGGGKVRMKF YVWK -NH2 Genepep, France N/A Bi4N1K peptide (with PEG spacer) Ac-KRFYVVMWKK-PEG7-KRFYVVMWKK-NH2 PEG7 = NH2-(CH2CH2O)7-CH2CH2COOH Genepep, France N/A Bi4N1K peptide (with 8G spacer) Ac-KRFYVVMWKKGGGGG GGGKRFYVVMWKK-NH2 Genepep, France N/A (Continued on next page) e1 Immunity 53, 429–441.e1–e8, August 18, 2020 .. REAGENT or RESOURCE SOURCE IDENTIFIER HTRA1 inhibitor DPMFKLboroV Roche Innovation Center, Basel, Swiss www.roche.com Critical Commercial Assays EasySep Mouse Monocyte Isolation Kit STEMCELL Technologies Cat#19861 EasySep Human Monocyte Enrichment Kit STEMCELL Technologies Cat#19059 Human HTRA1 Elisa Elabscience Cat#E-EL-H0423 Humain OPN Elisa R&D Systems Cat#DOST00 MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panels Millipore Cat#HCYTOMAG Experimental Models: Cell Lines COS-7 cell line ATCC Cat#CRL-1651, RRID: CVCL_0224 Experimental Models: Organisms/Strains Mouse: C57BL/6J (rd8 mutation free) The Jackson Laboratory RRID: IMSR_JAX:000664 Mouse: Opn / (rd8 mutation free) The Jackson Laboratory B6.129S6(Cg)-Spp1tm1Blh/J Stock No: 004936 Mouse: Thbs1 / (rd8 mutation free) The Jackson Laboratory B6.129S2-Thbs1tm1Hyn/J Stock No: 006141 Oligonucleotides Taqman primers for HTRA1 Thermo Fisher Scientific Cat#Hs01016151_m1 Taqman primers for ARMS2 Thermo Fisher Scientific Cat#Hs01394203_m1 Primers for human ARMS2 ARMS2 forward 5¢-TCGGTGGTT CCTGTGTCCTTCATT-3¢, ARMS2 reverse 5¢-TCACCTTGCTGCAG TGTGGATGA-3¢ Integrated DNA Technologies, Inc N/A Primers for human OPNSPP1 forward 5¢-GCACAGGTGATGC CTAGGAG-3¢, SPP1 reverse 5¢- ACCAGACTCGTCTCAGGAG-3¢ Integrated DNA Technologies, Inc N/A Primers for human PLEKHA1 PLEKHA1 forward 5¢-GCCCTGAA GAGATGCACAGT-3¢, PLEKHA1 reverse 5¢-AAGGGTTCGACAGCCTTCTG-3¢ Integrated DNA Technologies, Inc N/A Primers for Mus musculus Opn Spp1 forward 5¢-TCTTCTGCGG CAGGCATT-3¢Spp1 reverse 5¢-TGC CAATCTCATGGTCGT-3¢ Integrated DNA Technologies, Inc N/A Recombinant DNA Plasmid: pCX_CD47-V5; human CD47 with a C-terminal V5 flag This paper Genecust, Boynes, France N/A Plasmid: pCX_CD47-Myc; human CD47 with a C-terminal Myc flag This paper Genecust, Boynes, France N/A Software and Algorithms ProteinScape 2.1 Bruker Daltonics www.bruker.com GraphPad Prism GraphPad Software www.graphpad.com/ Compute pI/Mw Expasy https://web.expasy.org/compute_pi/ Data Analysis 3.4 Bruker www.bruker.com Mascot Server 2.2.07 Matrix Science www.matrixscience.com/ PEAKS Studio 8.5 Bioinformatics Solutions Inc. www.bioinfor.com MetaMorph Molecular Devices www.moleculardevices.com HCS iDev Cell Analysis Software Thermo Fisher Scientific Cat#SX000041A (Continued on next page) Immunity 53, 429–441.e1–e8, August 18, 2020 e2
Control:Article Title: The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-HTRA1 Abcam Cat#ab38611; RRID: AB_733053 Goat polyclonal anti-IBA1 Abcam Cat#ab5076; RRID: AB_2224402 Goat polyclonal anti-TSP1 Abcam Cat#ab111230; RRID: AB_10867429 Monoclonal anti-FLAG M2 antibody Sigma-Aldrich Cat#F3165; RRID: AB_259529 Duolink In Situ PLA Probe anti-Rabbit PLUS antibody Sigma-Aldrich Cat#DUO92002; RRID: AB_2810940 Duolink In Situ PLA Probe anti-Mouse MINUS Antibody Sigma-Aldrich Cat#DUO92004; RRID: AB_2713942 Mouse Monoclonal anti-V5 Tag Thermo Fisher Scientific Cat#R960-25; RRID: AB_2556564 Rabbit polyclonal anti-Myc tag Abcam Cat#ab9106; RRID: AB_307014 Mouse monoclonal anti-CD36 Abcam Cat#ab17044; RRID: AB_443600 Mouse monoclonal anti-CD47 Abcam Cat#ab3283; RRID: AB_303671 Mouse monoclonal anti-TSP1 Thermo Fisher Scientific Cat#MA5-13390; RRID: AB_10979326 Mouse IgG1 Isotype Control R&D Systems Cat#MAB002; RRID: AB_357344 Goat IgG1 Isotype Control R&D Systems Cat#AB-108-C; RRID: AB_354267 Rabbit polyclonal anti-PU.1 Thermo Fisher Scientific Cat#PA5-17505; RRID: AB_10989141 Goat polyclonal anti-Otx2 R&D Systems Cat#AF1979; RRID: AB_2157172 Goat polyclonal anti-mouse Osteopontin R&D Systems Cat#AF808; RRID: AB_2194992 Goat polyclonal anti-human Osteopontin R&D Systems Cat#AF1433; RRID: AB_354791 Rabbit polyclonal anti-Iba1 Wako Cat#019-19741; RRID: AB_839504 Rat monoclonal anti-CD102 BD Biosciences Cat#553325; RRID: AB_394783 Biological Samples Peripheral blood from human donors CHNO des Quinze- Vingts, Paris, France N/A Post-mortem RPE/choroid/sclera and retinal pieces from healthy and AMD donors Minnesota Lions Eye Bank, USA N/A Healthy porcine eyes Guy Harang slaughterhouse, Houdan, France N/A Chemicals, Peptides, and Recombinant Proteins Recombinant Human Thrombospondin-1 Protein R&D Systems Cat3074-TH-050 Recombinant Human Thrombospondin-1 C-terminal domain (Cter) with a FLAG-epitope at the N terminus GTP Technology, Labege, France N/A Recombinant Human HTRA1 Protein R&D Systems Cat#2916-SE-020 Control peptide (KRFYGGMWKK) Genepep, France N/A 4N1K CD47-activating peptide (KRFYVVMWKK) Genepep, France N/A Control bi- peptide Ac- KVRMKF YVWKGGGGGGGGKVRMKF YVWK -NH2 Genepep, France N/A Bi4N1K peptide (with PEG spacer) Ac-KRFYVVMWKK-PEG7-KRFYVVMWKK-NH2 PEG7 = NH2-(CH2CH2O)7-CH2CH2COOH Genepep, France N/A Bi4N1K peptide (with 8G spacer) Ac-KRFYVVMWKKGGGGG GGGKRFYVVMWKK-NH2 Genepep, France N/A (Continued on next page) e1 Immunity 53, 429–441.e1–e8, August 18, 2020 .. REAGENT or RESOURCE SOURCE IDENTIFIER HTRA1 inhibitor DPMFKLboroV Roche Innovation Center, Basel, Swiss www.roche.com Critical Commercial Assays EasySep Mouse Monocyte Isolation Kit STEMCELL Technologies Cat#19861 EasySep Human Monocyte Enrichment Kit STEMCELL Technologies Cat#19059 Human HTRA1 Elisa Elabscience Cat#E-EL-H0423 Humain OPN Elisa R&D Systems Cat#DOST00 MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panels Millipore Cat#HCYTOMAG Experimental Models: Cell Lines COS-7 cell line ATCC Cat#CRL-1651, RRID: CVCL_0224 Experimental Models: Organisms/Strains Mouse: C57BL/6J (rd8 mutation free) The Jackson Laboratory RRID: IMSR_JAX:000664 Mouse: Opn / (rd8 mutation free) The Jackson Laboratory B6.129S6(Cg)-Spp1tm1Blh/J Stock No: 004936 Mouse: Thbs1 / (rd8 mutation free) The Jackson Laboratory B6.129S2-Thbs1tm1Hyn/J Stock No: 006141 Oligonucleotides Taqman primers for HTRA1 Thermo Fisher Scientific Cat#Hs01016151_m1 Taqman primers for ARMS2 Thermo Fisher Scientific Cat#Hs01394203_m1 Primers for human ARMS2 ARMS2 forward 5¢-TCGGTGGTT CCTGTGTCCTTCATT-3¢, ARMS2 reverse 5¢-TCACCTTGCTGCAG TGTGGATGA-3¢ Integrated DNA Technologies, Inc N/A Primers for human OPNSPP1 forward 5¢-GCACAGGTGATGC CTAGGAG-3¢, SPP1 reverse 5¢- ACCAGACTCGTCTCAGGAG-3¢ Integrated DNA Technologies, Inc N/A Primers for human PLEKHA1 PLEKHA1 forward 5¢-GCCCTGAA GAGATGCACAGT-3¢, PLEKHA1 reverse 5¢-AAGGGTTCGACAGCCTTCTG-3¢ Integrated DNA Technologies, Inc N/A Primers for Mus musculus Opn Spp1 forward 5¢-TCTTCTGCGG CAGGCATT-3¢Spp1 reverse 5¢-TGC CAATCTCATGGTCGT-3¢ Integrated DNA Technologies, Inc N/A Recombinant DNA Plasmid: pCX_CD47-V5; human CD47 with a C-terminal V5 flag This paper Genecust, Boynes, France N/A Plasmid: pCX_CD47-Myc; human CD47 with a C-terminal Myc flag This paper Genecust, Boynes, France N/A Software and Algorithms ProteinScape 2.1 Bruker Daltonics www.bruker.com GraphPad Prism GraphPad Software www.graphpad.com/ Compute pI/Mw Expasy https://web.expasy.org/compute_pi/ Data Analysis 3.4 Bruker www.bruker.com Mascot Server 2.2.07 Matrix Science www.matrixscience.com/ PEAKS Studio 8.5 Bioinformatics Solutions Inc. www.bioinfor.com MetaMorph Molecular Devices www.moleculardevices.com HCS iDev Cell Analysis Software Thermo Fisher Scientific Cat#SX000041A (Continued on next page) Immunity 53, 429–441.e1–e8, August 18, 2020 e2
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