Incubation:Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment
Article Snippet: .. Subsequently, the slides were incubated overnight at 4 °C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or FOXP3 (1:1500, UM870140, Origene). .. Following primary antibody incubation, the sections were treated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG H&L secondary antibody (bs-0295G-HRP, Bioss) for 1 h at room temperature.
Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment.
Article Snippet: .. Subsequently, the slides were incubated overnight at 4°C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or AR TI CL E IN P RE SS FOXP3 (1:1500, UM870140, Origene). .. Following primary antibody incubation, the sections were treated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG H&L secondary antibody (bs-0295G-HRP, Bioss) for 1 hour at room temperature.
Article Title: GITRL impairs hepatocyte repopulation by liver progenitor cells to aggravate inflammation and fibrosis by GITR + CD8 + T lymphocytes in CDE Mice
Article Snippet: .. The sections were incubated with goat anti-TdTomato (1:400, Biorbyt, Catalogue Number: orb182397), mouse anti-cytokertin 19 (CK19) (1:200, RnD Systems, Catalogue Number: MAB3506), mouse anti-hepatocyte nuclear factor (HNF)4α (1:200, Abcam, Catalogue Number: ab41898), or rabbit anti-GITRL (1:150, ProteinTech, Catalogue Number: 23899-1-AP) primary antibodies. .. For visualization, the tissue sections were incubated with secondary antibodies conjugated to Alexa 488 (1:600; Molecular Probes, Catalogue Number: A-21202 or A-21206) and/or Alexa 555 (1:600; Molecular Probes, Catalogue Number: A-21432 or A-31572) under a Nikon 50i fluorescence microscope (Nikon, Japan) and scanned by a Panoramic MIDI Digital Slide Scanner (3DHISTECH, HUN).
Recombinant:Article Title: Microglia mediated by LAG3 regulate astrocyte morphogenesis in the brain white matter.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat Anti-IBA1 Abcam Cat# ab5076; RRID: AB_2224402 Rabbit Anti-IBA1 Wako Cat# 019-19741; RRID: AB_839504 Rabbit Anti-LAG3 Millipore Cat# MABF954; RRID: AB_3073509 Rat Anti-CD68 Bio-Rad Cat# MCA1957; RRID: AB_2335597 Rabbit Anti-CD68 Proteintech Cat# 28058-1-AP; RRID: AB_2881049 Rabbit Anti-CX3CR1 Abcam Cat# ab8021; RRID: AB_306203 Rabbit Anti-Ki67 Abcam Cat# ab15580; RRID: AB_443209 Rabbit Anti-GFAP Agilent Cat# Z0334; RRID: AB_10013382 Mouse Anti-GFAP Sigma Cat# G6171; RRID: AB_1840893 Mouse Anti-ALDH1L1 Abcam Cat# ab56777; RRID: AB_940204 Mouse Anti-ACSBG1 Novus Biologicals Cat# NBP2-02554; RRID: AB_3245453 Rabbit Anti-Olig2 Abcam Cat# ab109186; RRID: AB_10861310 Mouse Anti-L1CAM Abcam Cat# ab24345; RRID: AB_448025 Rabbit Anti-p-P38 MAPK Cell Signaling Technology Cat# 4511P; RRID: AB_2139682 Rabbit Anti-P38 MAPK Cell Signaling Technology Cat# 8690S; RRID: AB_10999090 Rabbit Anti-AKT Abmart Cat# T55561F; RRID: AB_2936969 Rabbit Anti-p-AKT Abmart Cat# T40067F; RRID: AB_2936953 Rabbit Anti-mTOR Cell Signaling Technology Cat# 2983T; RRID: AB_2105622 Rabbit Anti-p-mTOR Abmart Cat# T56571F; RRID: AB_2936395 Mouse Anti-β-Actin Proteintech Cat# 60008-1-Ig; RRID: AB_2289225 Rabbit Anti-β-Actin Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Mouse Anti-Flag Sigma-Aldrich Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Cell Signaling Technology Cat# 3724S; RRID: AB_1549585 Rabbit Anti-GITRL Proteintech Cat# 23899-1-AP; RRID: AB_2879355 Rabbit Anti-GITR Cell Signaling Technology Cat# 37472T; RRID:AB_3094648 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Anisomycin MedChemExpress Cat# HY-18982 Rapamycin MedChemExpress Cat# HY-10219 Torin1 MedChemExpress Cat# HY-13003 Recombinant Murine GITRL MedChemExpress Cat# HY-P7319 Critical commercial assays RNAprep Pure Micro Kit TIANGEN DP420 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE277980 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 Experimental models: Organisms/strains Mouse: Lag3 fl/ Shanghai Model Organisms Center N/A Mouse: Cx3cr1-Cre Jackson laboratory JAX: 025524 Oligonucleotides Primers for QPCR, see method details This paper N/A (Continued on next page) 18 Cell Reports 44, 116112, August 26, 2025 .. Cx3cr1-Cre mice (JAX stock # 025524: B6J.B6N(Cg)-Cx3cr1 tm1.1(cre)Jung /J), Cxcr1-GFP mice (JAX stock # 005582: B6.129P2(Cg)Cx3cr1 tm1Litt /J) and Cx3cr1-CreER mice (JAX stock # 021160) were obtained from the Jackson Laboratory.
RNA Sequencing:Article Title: Microglia mediated by LAG3 regulate astrocyte morphogenesis in the brain white matter.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat Anti-IBA1 Abcam Cat# ab5076; RRID: AB_2224402 Rabbit Anti-IBA1 Wako Cat# 019-19741; RRID: AB_839504 Rabbit Anti-LAG3 Millipore Cat# MABF954; RRID: AB_3073509 Rat Anti-CD68 Bio-Rad Cat# MCA1957; RRID: AB_2335597 Rabbit Anti-CD68 Proteintech Cat# 28058-1-AP; RRID: AB_2881049 Rabbit Anti-CX3CR1 Abcam Cat# ab8021; RRID: AB_306203 Rabbit Anti-Ki67 Abcam Cat# ab15580; RRID: AB_443209 Rabbit Anti-GFAP Agilent Cat# Z0334; RRID: AB_10013382 Mouse Anti-GFAP Sigma Cat# G6171; RRID: AB_1840893 Mouse Anti-ALDH1L1 Abcam Cat# ab56777; RRID: AB_940204 Mouse Anti-ACSBG1 Novus Biologicals Cat# NBP2-02554; RRID: AB_3245453 Rabbit Anti-Olig2 Abcam Cat# ab109186; RRID: AB_10861310 Mouse Anti-L1CAM Abcam Cat# ab24345; RRID: AB_448025 Rabbit Anti-p-P38 MAPK Cell Signaling Technology Cat# 4511P; RRID: AB_2139682 Rabbit Anti-P38 MAPK Cell Signaling Technology Cat# 8690S; RRID: AB_10999090 Rabbit Anti-AKT Abmart Cat# T55561F; RRID: AB_2936969 Rabbit Anti-p-AKT Abmart Cat# T40067F; RRID: AB_2936953 Rabbit Anti-mTOR Cell Signaling Technology Cat# 2983T; RRID: AB_2105622 Rabbit Anti-p-mTOR Abmart Cat# T56571F; RRID: AB_2936395 Mouse Anti-β-Actin Proteintech Cat# 60008-1-Ig; RRID: AB_2289225 Rabbit Anti-β-Actin Proteintech Cat# 20536-1-AP; RRID: AB_10700003 Mouse Anti-Flag Sigma-Aldrich Cat# F1804; RRID: AB_262044 Rabbit Anti-HA Cell Signaling Technology Cat# 3724S; RRID: AB_1549585 Rabbit Anti-GITRL Proteintech Cat# 23899-1-AP; RRID: AB_2879355 Rabbit Anti-GITR Cell Signaling Technology Cat# 37472T; RRID:AB_3094648 Chemicals, peptides, and recombinant proteins Papain Worthington Cat# 38P18865 Anisomycin MedChemExpress Cat# HY-18982 Rapamycin MedChemExpress Cat# HY-10219 Torin1 MedChemExpress Cat# HY-13003 Recombinant Murine GITRL MedChemExpress Cat# HY-P7319 Critical commercial assays RNAprep Pure Micro Kit TIANGEN DP420 Deposited data Raw and analyzed data of bulk RNA-seq This paper GEO: GSE277980 Experimental models: Cell lines HEK293 Cell Line ATCC ATCC CRL-1573; RRID: CVCL_0045 Experimental models: Organisms/strains Mouse: Lag3 fl/ Shanghai Model Organisms Center N/A Mouse: Cx3cr1-Cre Jackson laboratory JAX: 025524 Oligonucleotides Primers for QPCR, see method details This paper N/A (Continued on next page) 18 Cell Reports 44, 116112, August 26, 2025 .. Cx3cr1-Cre mice (JAX stock # 025524: B6J.B6N(Cg)-Cx3cr1 tm1.1(cre)Jung /J), Cxcr1-GFP mice (JAX stock # 005582: B6.129P2(Cg)Cx3cr1 tm1Litt /J) and Cx3cr1-CreER mice (JAX stock # 021160) were obtained from the Jackson Laboratory.
Immunofluorescence:
Article Title: GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt.
Article Snippet: Serial Z-stack sectioning was done at Cell Death and Disease (2022) 13:297 125 nm of intervals and tacks of SIM images were reconstructed using softWoRx 6.1.1 (Cytiva, GE Healthcare). .. For immune-colocalization analysis, the cultured cells were fixed with 4% paraformaldehyde and permeabilized for standard intracellular immunofluorescence staining as described previously [54] to detect GITRL (ProteinTech) and ANXA2 (Cell Signaling Technology) by a Nikon A1 confocal microscope (Nikon). .. For flow cytometry analysis, the cultured cells were digested by trypsin and fixed with 4% paraformaldehyde for surface immunofluorescence staining without permeabilization to detect the expression of GITRL (ProteinTech) and GITR (Abcam, Cambridge, UK) or for standard intracellular immunofluorescence staining to detect the expression of ANXA2 (Cell Signaling Technology) by FACSCalibur flow cytometry (BD Biosciences) using CellQuest software (BD Biosciences) as described previously [51].
Cell Culture:
Article Title: GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt.
Article Snippet: Serial Z-stack sectioning was done at Cell Death and Disease (2022) 13:297 125 nm of intervals and tacks of SIM images were reconstructed using softWoRx 6.1.1 (Cytiva, GE Healthcare). .. For immune-colocalization analysis, the cultured cells were fixed with 4% paraformaldehyde and permeabilized for standard intracellular immunofluorescence staining as described previously [54] to detect GITRL (ProteinTech) and ANXA2 (Cell Signaling Technology) by a Nikon A1 confocal microscope (Nikon). .. For flow cytometry analysis, the cultured cells were digested by trypsin and fixed with 4% paraformaldehyde for surface immunofluorescence staining without permeabilization to detect the expression of GITRL (ProteinTech) and GITR (Abcam, Cambridge, UK) or for standard intracellular immunofluorescence staining to detect the expression of ANXA2 (Cell Signaling Technology) by FACSCalibur flow cytometry (BD Biosciences) using CellQuest software (BD Biosciences) as described previously [51].
Staining:
Article Title: GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt.
Article Snippet: Serial Z-stack sectioning was done at Cell Death and Disease (2022) 13:297 125 nm of intervals and tacks of SIM images were reconstructed using softWoRx 6.1.1 (Cytiva, GE Healthcare). .. For immune-colocalization analysis, the cultured cells were fixed with 4% paraformaldehyde and permeabilized for standard intracellular immunofluorescence staining as described previously [54] to detect GITRL (ProteinTech) and ANXA2 (Cell Signaling Technology) by a Nikon A1 confocal microscope (Nikon). .. For flow cytometry analysis, the cultured cells were digested by trypsin and fixed with 4% paraformaldehyde for surface immunofluorescence staining without permeabilization to detect the expression of GITRL (ProteinTech) and GITR (Abcam, Cambridge, UK) or for standard intracellular immunofluorescence staining to detect the expression of ANXA2 (Cell Signaling Technology) by FACSCalibur flow cytometry (BD Biosciences) using CellQuest software (BD Biosciences) as described previously [51].
Microscopy:Article Title: GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt.
Article Snippet: Serial Z-stack sectioning was done at Cell Death and Disease (2022) 13:297 125 nm of intervals and tacks of SIM images were reconstructed using softWoRx 6.1.1 (Cytiva, GE Healthcare). .. For immune-colocalization analysis, the cultured cells were fixed with 4% paraformaldehyde and permeabilized for standard intracellular immunofluorescence staining as described previously [54] to detect GITRL (ProteinTech) and ANXA2 (Cell Signaling Technology) by a Nikon A1 confocal microscope (Nikon). .. For flow cytometry analysis, the cultured cells were digested by trypsin and fixed with 4% paraformaldehyde for surface immunofluorescence staining without permeabilization to detect the expression of GITRL (ProteinTech) and GITR (Abcam, Cambridge, UK) or for standard intracellular immunofluorescence staining to detect the expression of ANXA2 (Cell Signaling Technology) by FACSCalibur flow cytometry (BD Biosciences) using CellQuest software (BD Biosciences) as described previously [51].
|