Staining:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
SDS Page:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
Bicinchoninic Acid Protein Assay:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
cDNA Synthesis:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
Reverse Transcription:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
SYBR Green Assay:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
Fractionation:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
Immunoprecipitation:Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.
Article Snippet: Biological samples Human: NF1 patient’s tumor tissues Emory University Approval number # STUDY00002544 Mouse: Retina samples from Nf1+/− , Nf1f/f and Nf1ΔMΦ mice This paper N/A Mouse: bone marrow-derived macrophages from Nf1f/f and Nf1ΔMΦ mice This paper N/A Chemicals, peptides, and recombinant proteins Macrophage colony stimulating factor (M-CSF) Invitrogen Cat# PMC2044 Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich Cat# P1585 Lipopolysaccharide (LPS) Selleckchem Cat# S7850 Murine IFN-gamma (IFNγ) PeproTech Cat# 315-05 Murine IL-4 PeproTech Cat# 214-14 Akt-2 inhibitor (CCT128930; Akt2i) Selleckchem Cat# S2635 GLUT1 inhibitor; BAY-876 inhibitor Cayman Chemical Company Cat# 19961 FcR (CD16/CD32) Blocking reagent BD Biosciences Cat# 553141 BD Cytofix/Cytoperm buffer BD Biosciences Cat# 555028 BD Perm/Wash buffer BD Biosciences Cat# 555028 (Continued on next page) Cell Reports 44, 115625, May 27, 2025 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025 ..
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