irf1 rabbit antibody  (Proteintech)

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a GSEA using GO pathways was performed between shAPC or scramble control transfected CT26 subcutaneous tumors, using gene sets associated with type II interferon response and antigen processing and presentation. b Pathway responsive genes for activity inference from gene expression (progeny) analysis performed between tumors formed in Apc-silenced and control groups. c Total cell lysates from a series of IFNγ concentrations were subjected to immunoblot analysis with antibodies to the indicated proteins. Data represent three independent experiments. d Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression (RT-qPCR) in CT26-shApc or CT26-scramble cells. n = 6 per group, one-way ANOVA. e Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. f MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. g Numbers of OVA-tetramer positive CD8 + T cells in TILs of MC38-OVA-shAPC subcutaneous tumors as detected by flow cytometry. n = 3 for each group, one-way ANOVA. h Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in the indicated cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. i APC-silenced CT26 cells were transfected with Stat1 R274Q and Irf1 overexpression lentivirus, then subcutaneously xenotransplanted to Balb/c mice; tumor growth was monitored at the indicated times. n = 8 for each group, two-way ANOVA. j Scatterplot showing numbers of CD8 + cells in the indicated groups. n = 8, 7, 8 for each group, one-way ANOVA. k AKP organoids were transfected with Stat1 R274Q and Irf1 overexpression lentivirus, then orthotopically inoculated into C57BL/6 mice; tumor growth was monitored and scored by colonoscopy. n = 6 for each group, one-way ANOVA. l Representative immunofluorescence staining of CK (red) and CD8 (yellow) in tumor tissues, with scatterplot showing numbers of CD8 + cells in three groups. n = 6 for each group, one-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Control, Transfection, Activity Assay, Gene Expression, Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Over Expression, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Apc-silenced CT26 cells transfected with negative control (N.C) or two single guide RNAs (sgRNAs) targeting Ptpn13 were stimulated with different concentrations of IFNγ. Total cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins. Data are representative of three independent experiments. b Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in Ptpn13-knockout or negative control sgRNA-transfected CT26-shAPC cells with 12 h exposure to IFNγ (50 ng/mL). n = 6 per group, one-way ANOVA. c Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. d Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. e Numbers of OVA-tetramer positive CD8 + T cells in TILs of Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC subcutaneous tumors, as detected by flow cytometry. n = 3 for each group, one-way ANOVA. f Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in intestinal tumors of TAM- or oil-treated APV mice. n = 6 per group, one-way ANOVA. g MFI of H-2Kb/2Db positive cells in intestinal tumors of TAM- or oil-treated APV mice as detected by flow cytometry. n = 6 per group, unpaired t -test. h Scatterplot showing correlation between MFI of HLA-ABC and Ptpn13 IF staining in CRC primary tissues. n = 80, Pearson’s r . i, j CRC-patient-derived organoids (PDO) were cultivated and transfected with Ptpn13-knockout or negative control sgRNA. i Representative immunofluorescence staining of Epcam (green), HLA-ABC (red) and CD8 (cyan). j MFI of HLA-ABC IF staining in Ptpn13-knockout or negative control sgRNA-transfected PDOs. One-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Knock-Out, Flow Cytometry, Staining, Derivative Assay, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Flag-tagged Stat1 expression vector (0.5 μg) was transfected into CT26 cells. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. b Schematic diagram showing the GST-fused PTPN13 motifs and His-tagged STAT1 used in the GST pull-down assays. c GST pull-down assays examining the interactions between GST-fused PTPN13 fragments and His-tagged STAT1 protein. Data are representative of three independent experiments. d Phosphorylated STAT1 was immunoprecipitated with anti-Flag from IFNγ-stimulated 293T transfectants and incubated with 0.2 mg/mL recombinant GST-PTPase domain of PTPN13. Immunoprecipitates were immunoblotted with anti-phospho-STAT1. Equal loading was verified by reprobing with anti-STAT1. e Total cell lysates of CT26 cells were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. f CT26 cells were treated with the indicated concentrations of IFNγ, and cell lysates were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. g A Flag-tagged Stat1 vector (0.5 μg) was transfected into CT26-shApc cells or their control cells. Total cell lysates from the indicated cells were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. h Alphafold3-predicted binding pattern of human APC (brown) and the PDZ2a domain of PTPN13 (cyan). The APC V2843 residue is labeled, and APC Q2829–V2843 residues are shown as sticks and colored in yellow. Hydrogen bonds are shown as yellow dotted lines. i CT26 cells were transfected with HA-tagged Apc-WT or Apc V2860A mutant plasmids, and total cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-PTPN13 and anti-CTNNB1. Data are representative of three independent experiments. j IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. k CRISPR/Cas9-based establishment of APC V2860A point mutation. l CT26 cells transfected with APC-WT or APC V2860A mutant plasmids (CT26-APC V2860A -1/2) were incubated with or without IFNγ at the indicated concentrations for 2 h, and total cell lysates were subjected to immunoblot analysis. m IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids (CT26-APC V2860A -1/2) for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. n CT26 cells harboring gRNA-induced mutant APC V2860A (CT26-APC V2860A -1/2) and the WT control were subcutaneously injected (2 × 10 6 cells) into Balb/c mice, and tumor growth was monitored. n = 8 for each group, two-way ANOVA. o Immunofluorescence of CD8 + cell infiltration in subcutaneous tumors of CT26-APC V2860A and the control group. n = 8 for each group, one-way ANOVA. Data are representative of three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Incubation, Recombinant, Control, Binding Assay, Residue, Labeling, Mutagenesis, Quantitative RT-PCR, CRISPR, Western Blot, Injection, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Schematic diagram showing the indicated residues at the APC C terminus. b Kinetics of the interaction between PDZ-2a and the indicated residues of APC were explored by surface plasmon resonance-based binding assays. c Binding affinities of PDZ2a to APC C-terminal peptides of different lengths, as measured by an FP assay. d The 2.1-Å complex structure of the PDZ2a domain (1364–1446 aa) and the APC11 peptide. PDZ2a is shown in cyan and presented as a surface diagram, whereas the peptide is shown in yellow and presented as a stick diagram. e Detailed interactions between the APC11 peptide and PDZ2a within the complex. The PDZ2a residues involved are labeled and shown as magenta sticks, and the peptide-interacting water molecules are shown as green balls. Hydrogen bonds are shown as yellow dotted lines. f Binding affinity of PDZ2a to the WT APC11 peptide and the APC11M mutant (V2843A) as measured by an FP assay. g GST-fused STAT1 was incubated with HA-tagged PDZ2a and with TAT-APC11 or TAT-APC11M peptides, immunoprecipitated with GST beads, and immunoblotted with anti-GST and anti-HA antibodies. Data are representative of three independent experiments. h CT26 cells transfected with a Flag-tagged Stat1 vector were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 4 h. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. i Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. j Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in Apc-silenced CT26 cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. k Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of the MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kd/2Dd antibody or isotype control antibodies. Data were calculated from three independent experiments. One-way ANOVA. l Apc-silenced MC38-OVA 257-264 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of OVA 257-264 -specific MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kb/SIINFEKL antibody or isotype control antibodies. Data represent three independent experiments. One-way ANOVA. m DLD1 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. n IRF1, LMP2, TAP1, TAP2, HLA-A, HLA-B, HLA-C , and B2M mRNA expression (RT-qPCR) in the indicated cells after exposure to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: SPR Assay, Binding Assay, FP Assay, Labeling, Mutagenesis, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Staining, Control

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a Violin plot displaying the expression level of top transcription factors (TFs) in luminal subtypes at −4W and +1W. b Heatmap showing the regulon activities of the top TFs in luminal subtypes at −4W. c Immunohistochemical staining for IRF1 in the goat mammary gland at −4W and +1W. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. d Quantification of IRF1-positive cells in c . n = 8 sections from 4 goats. e Representative images of immunohistochemical staining for PR in the goat mammary organoids treated with or without IFNγ. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. f Quantification of PR-positive cells in e . n = 5 domes per group. g Representative images of carmine-stained mammary gland whole mounts in WT and IRF1-KO mice at 9 weeks. Scale bars, 0.4 mm. h − k Automatic quantification of the number junctions ( h ), tips ( i ), branches ( j ) and lumen diameters ( k ) of mammary tissues in f . n = 6 mice in wild type and n = 3 in IRF1-KO mice. n = 30 and n = 15 ductal lumens in WT and IRF1-KO mice, respectively. l , m Immunohistochemical staining ( l ) and quantification ( m ) of PR and ER in mammary tissues from WT or IRF1-KO mice at 9 weeks. Nuclei were counterstained with hematoxylin ( l ). n = 4 mice per group. Scale bars, 10 μm. n , o Immunohistochemical staining ( n ) and quantification ( o ) of PR and ER in mammary tissues from WT or IRF1-KO mice during RR. Nuclei were counterstained with hematoxylin. n = 5 mice per group. Scale bars, 10 μm. p Pre-ranked GSEA graphical output for the enrichment in IRF1-KO mice mammary glands of the gene set estrogen response early from the Molecular Signatures Database Hallmarks collection. n = 3 mice per group. q Heatmap representing the log 2 fold change expression of hormone-driven genes in IRF-KO compared to WT at 9 weeks. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in d , f , h – k , m , o .

Article Snippet: Bead-bound nuclei were then incubated overnight at 4 °C with IRF1 rabbit antibody (1:100 dilution, 11335-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a The genomic loci with IRF1 motifs are selected and the ATAC-seq signal intensity is shown in heatmaps. The average signal intensity is shown on top. b Heatmap displaying the transcriptional level of genes presumably bound by IRF1. n = 3 goats per group. c Heatmaps showing the signal intensity of IRF1 CUT&Tag in goat mammary tissues at −4W and +1 W. The average signal intensity is shown on top. d ATAC-seq and IRF1 CUT&Tag profiles at the ESRRB locus in −4W and +1 W are shown. The differential regions between −4W and +1 W with IRF1 motifs are highlighted in yellow. e UMAP plot showing the specific expression of ESRRB in LumHR cells by scRNA-seq data. f , g Immunohistochemical staining and quantification of ESRRB in goat mammary tissues at −4W and +1 W. Representative images of Immunohistochemical staining ( f ). Nuclei are counterstained with hematoxylin. n = 10 sections from 5 goats per group. Scale bars, 20 μm. Two-sided Student’s t -test. h Luciferase reporter assays in goat mammary epithelial cells. Cells are transfected with WT IRF1 motif (IRF1-MWT) or IRF1-motif site mutation (IRF1-MM) vector and treated with IFNγ or not. n = 4 biological replicates. Two-way ANOVA test. i , j Immunohistochemical staining and quantification of ESRRB in mouse WT or IRF1-KO mammary tissues under RR. Representative images of Immunohistochemical staining ( i ). Nuclei are counterstained with hematoxylin. n = 4 mice per group. Scale bars, 50 μm. Two-sided Student’s t -test. k The proposed model in the current study is that a reduction of LumHR cells triggered by IRF1-ESRRB signaling upregulation promotes the accumulation of LumSecP during RR in ruminants. LumHR cells control the differentiation of LumSecP to LumSec cells through the PRLR pathway and regulate the cell composition of luminal lineages during RR. Created with BioRender.com.

Article Snippet: Bead-bound nuclei were then incubated overnight at 4 °C with IRF1 rabbit antibody (1:100 dilution, 11335-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining, Luciferase, Transfection, Mutagenesis, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: Graphical abstract. Graphical abstract illustrating the hypothetical mechanism by which JMJD6 promotes tumor progression and immune evasion in GC. JMJD6 is overexpressed in gastric cancer cells and promotes BRD4 expression, which upregulates IRF1 and consequently increases PD-L1 expression. Elevated PD-L1 expression on tumor cells inhibits T cell–mediated antitumor immunity, thereby facilitating immune escape.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: JMJD6 regulates BRD4, IRF1 and PD-L1 expression. ( a ) The knockdown of JMJD6 by transfection with siRNA-JMJD6 suppressed BRD4, IRF1 and PD-L1 in MKN74. In addition, the knockdown of BRD4 by transfection with siRNA-BRD4 suppressed IRF1 and PD-L1 in MKN74. In contrast, the knockdown of BRD4 did not suppress JMJD6 in MKN74. ( b ) Knockdown of JMJD6 suppressed PD-L1 and BRD4 expression in MKN74 gastric cancer (GC) cells. White dotted lines indicate nuclear boundaries. ( c ) Co-culture assay of GC cells and T cells. Under JMJD6 knockdown, T cells had more potent anti-tumor activity against GC cells compared with NC, and the proliferation ratio of GC cells was significantly decreased (mean ± SD, n = 3; error bars indicate SD, n = 3). ( d ) An impedance-based tumor-cell killing assay. The knockdown of JMJD6 increased the anti-tumor activity of T cells and inhibited the proliferation of GC cells. ( e ) JMJD6 overexpression using plasmid transfection promotes BRD4, IRF1 and PD-L1 expression. ( f ) A hypothetical model of the overexpression or activation of JMJD6 in GC cells.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing, Knockdown, Transfection, Co-culture Assay, Activity Assay, Over Expression, Plasmid Preparation, Activation Assay

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: HSV-1 infection induces IRF1 and MITA/STING contributes to IRF1 induction. (A–B) HT1080 cells were either mock-infected or infected with HSV-1 (MOI = 5) for 8 h. The expression of IRF1 was quantified by RT-qPCR (A), and whole cell lysates (WCLs) were collected and analyzed by immunoblotting (B). (C–E) HT1080 cells transduced with control sgRNA (Ctrl) or sgRNA targeting MITA/STING were mock-infected or infected with HSV-1 (MOI = 5), and the expression of IRF1 (C) , IFNB1 (D), and CXCL10 (E) was quantified at 8 h post-infection. (F) HT1080 cells were mock-infected or infected with HSV-1 (MOI = 5), and WCLs were analyzed by immunoblotting at 8 h post-infection. (G–I) HT1080 cells were treated with H-151 (5 μM), and mock-infected or infected with HSV-1 (MOI = 5) for 8 h. The expression of IRF1 (G) , IFNB1 (H), and CXCL10 (I) was quantified by RT-qPCR. (J) HT1080 cells were treated with H-151 (5 μM), and mock-infected or infected with HSV-1 (MOI = 5) for 8 h. WCLs were analyzed by immunoblotting. (K–L) HT1080 cells transduced with control sgRNA (Ctrl) or sgRNA targeting MITA/STING. The expression of IRF1 was quantified by RT-qPCR (K), and WCLs were analyzed by immunoblotting (L).

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Transduction, Control

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: MITA activation is sufficient to induce IRF1 . (A) HT1080 cells were stimulated with diABZI (2.5 μM), and the expression of IRF1 , IFNB1 , ISG56, and CXCL10 was quantified by RT-qPCR at 6 h post-stimulation. (B) HT1080 cells were stimulated with diABZI (2.5 μM), and WCLs were analyzed by immunoblotting at 6 h post-stimulation. (C) THP-1 cells were stimulated with diABZI (2.5 μM), and the expression of IRF1 , IFNB1 , ISG56, and CXCL10 was quantified by RT-qPCR at 6 h post-stimulation. (D) THP-1 cells were stimulated with diABZI (2.5 μM), and WCLs were analyzed by immunoblotting at the indicated time points post-stimulation. (E) HT1080 cells were stimulated with MSA-2 (20 μM) or SR-717 (10 μM), and the expression of IRF1 , IFNB1 , ISG56, and CXCL10 was quantified by RT-qPCR at 8 h post-stimulation. (F) HT1080 cells were stimulated with MSA-2 (20 μM) or SR-717 (10 μM), and WCLs were analyzed by immunoblotting at 8 h post-stimulation.

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 restricts HSV-1 replication . (A) HT1080 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were infected with HSV-1 (MOI = 0.01). Viral titers in the supernatants were quantified at 48 h post-infection. (B–E) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 1). The expression of ICP0, ICP8, and UL19 was quantified by RT-qPCR (B–D). The mock-infected samples were labeled as N.D. (not detected), and the signals from wild-type (WT) cells infected with HSV-1 were normalized to 1. WCLs were analyzed by immunoblotting at 8 h post-infection (E). (F) HEK293T cells were transfected with an IFN-β promoter reporter plasmid mixture with increasing amounts of IRF1 expression plasmids (0, 0.1, 0.2, or 0.5 μg). Luciferase activities were measured at 24 h post-transfection. (G–H) HT1080 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were infected with HSV-1-GFP (MOI = 0.05), and GFP expression was imaged at 24 h post-infection (G). Scale bars,100 μm. Viral titers in the supernatants were quantified at 24 h post-infection (H).

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Transduction, Control, Infection, Expressing, Quantitative RT-PCR, Labeling, Western Blot, Transfection, Plasmid Preparation, Luciferase, Stable Transfection

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 amplifies HSV-1-triggered antiviral innate immunity. (A–B) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 5) for 6 h. RNA-seq was performed, and a Venn diagram displayed significantly upregulated ISG genes after HSV-1 infection (A). The human ISG gene set was obtained from previous studies ( ; ). Paired line plots showed the expression levels of these upregulated ISGs in control and IRF1 knockout cells (B). (C–I) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 5) for 6 h. The expression levels of the indicated genes were quantified by RT-qPCR.

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Transduction, Control, Infection, RNA Sequencing, Expressing, Knock-Out, Quantitative RT-PCR

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 interacts with IRF3 and promotes IRF3 recruitment to ISG promoters . (A) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 5), and WCLs were analyzed by immunoblotting at 8 h post-infection. (B) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 5), and nuclear and cytoplasmic fractions were isolated at the indicated time points and analyzed by immunoblotting. (C) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and precipitated samples were analyzed by immunoblotting. (D) HT1080 cells were infected with HSV-1 (MOI = 10) for 8 h. Co-immunoprecipitation was performed with the indicated antibodies, followed by immunoblotting analysis. (E) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10), and nuclear and cytoplasmic fractions were isolated at 8 h post-infection and analyzed by immunoblotting. (F) THP-1 cells were mock-infected or infected with HSV-1 (MOI = 10) for 5 or 10 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed with the indicated antibodies. (G) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed using an anti-IRF3 polyclonal antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (H) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were infected with HSV-1 (MOI = 10) for 10 h, followed by chromatin immunoprecipitation (ChIP) using an anti-IRF3 antibody or control IgG. IRF3 occupancy at the IFNB1 and IFNL1 promoter regions was assessed by qPCR.

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Transduction, Control, Infection, Western Blot, Isolation, Transfection, Immunoprecipitation, Labeling, Chromatin Immunoprecipitation

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 promotes antiviral innate immunity through its DNA-binding activity . (A) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and immunoprecipitated samples were analyzed by immunoblotting. (B–E) THP-1 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with HSV-1 (MOI = 5). The indicated genes were quantified by RT-qPCR (B–D), and WCLs were analyzed by immunoblotting at 8 h post-infection (E). (F) THP-1 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with HSV-1 (MOI = 10) for 8 h. Cell lysates were collected and pulldown assays were performed using a biotin-labeled ISG54 ISRE probe. The input and probe-bound proteins were analyzed by immunoblotting using an anti-IRF3 polyclonal antibody, and the input samples were also analyzed using an anti-IRF1 monoclonal antibody. (G–K) HT1080 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were mock-infected or infected with VSV (MOI = 5) for 8 h. The expression levels of the indicated genes were quantified by RT-qPCR.

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Binding Assay, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Control, Infection, Quantitative RT-PCR, Labeling

Journal: Nucleic Acids Research

Article Title: ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA

doi: 10.1093/nar/gkaf545

Figure Lengend Snippet: Identification of ERH and other novel positive regulators of IFNγ signaling by genome-wide genetic screening. ( A ) Schematic of the IFNγ-induced JAK/STAT signaling pathway, which stimulates expression of many genes, including IRF1. ( B ) Overview of FACS-based CRISPR-Cas9 knockout screen. Human RKO cells with dox-inducible iCas9 were transduced with a lentiviral genome-wide sgRNA library. Cas9 expression was induced for 2.5 or 5 days, after which cells were treated with IFNγ, and IRF1 induction detected by intracellular staining. Cells with the lowest or highest IRF1 levels were collected by FACS, and disrupted genes were identified by analyzing sgRNA-targeted coding sequences. ( C ) sgRNA enrichment in the IRF1 low cell population was plotted. Dashed lines indicate significance ( P ≤ 0.05) and enrichment (log2 fold change ≥ 1). Significantly enriched genes involved in the JAK/STAT pathway, the exon junction complex, or RNA splicing and export are highlighted. ( D ) Heatmap of selected IRF1 regulators as in ( C ) or MYC regulators involved in JAK/STAT signaling, the EJC, splicing and export, nonsense mediated decay, or type I interferon signaling. For each gene, the time point with the strongest enrichment is plotted. ( E ) RKO-iCas9 cells were transduced with vectors expressing the indicated sgRNAs. After 5 days of dox-induced Cas9 expression, cells were stimulated with IFNγ, after which endogenous IRF1 or MYC were detected by intra-cellular staining and flow cytometry. Representative samples from four (sg MAGOH ) or five (sg AAVS1 , sg ERH , and sg JAK2 ), n = 4 or 5 biological replicates, are shown. ( F ) Quantification of median fluorescence intensity (MFI) from panel E. Data represent means and sd; n = 4 or 5 biological replicates. One-way ANOVA with Bonferroni’s multiple comparison correction (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). ( G ) In parallel, IRF1 mRNA levels were measured by RT-qPCR. Data represent the mean and sd; n = 3 biological replicates. Two-tailed t-test with Benjamini–Hochberg correction (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).

Article Snippet: IRF1 levels were determined through intracellular staining with a PE-conjugated anti-IRF1 antibody (Cell Signaling Technology, 12 732).

Techniques: Genome Wide, Expressing, CRISPR, Knock-Out, Transduction, Staining, Flow Cytometry, Fluorescence, Comparison, Quantitative RT-PCR, Two Tailed Test