Journal: Veterinary Research

Article Title: Characterization of the rock pigeon type IV interferon system reveals conserved tyrosine-based signaling via the IFN-υR1/IL10RB complex

doi: 10.1186/s13567-026-01823-w

Figure Lengend Snippet: Sequence alignment of rock pigeon IFN-υR1 with IL10RB and other vertebrate homologs . Residue conservation is highlighted with black (identical), dark gray (highly similar), and light gray (low similarity). Predicted signal peptides and transmembrane domains are underlined. The D200 domain is marked by a solid line above the alignment. Boxes indicate potential intracellular binding sites for JAK1, TYK2, and STAT1. Reference sequences are as follows: zebrafish_IFN-υR1 ( NP_001035443.1 ); tropical clawed frog_IFN-υR1 ( XP_002940450.1 ); American alligator_IFN-υR1 ( XP_019337453.1 ); rock pigeon_IFN-υR1 ( XP_021145800.1 ); platypus_IFN-υR1 ( NW_001794458.1 : 8968090…8983283); zebrafish_IL10RB ( NP_001124447.1 ); tropical clawed frog_IL10RB ( NP_001165294.1 ); American alligator_IL10RB ( XP_059576675.1 ); rock pigeon_IL10RB ( XP_064922738.1 ); platypus_IL10RB ( XP_028937522.1 ).

Article Snippet: Commercial primary and secondary antibodies include Flag-Tag (Sigma-Aldrich, F1804, 1:6000 dilution), HA-Tag (Cell Signaling Technology, 3724S, 1:5000 dilution), STAT1 (ABclonal Technology, A19563, 1:5500 dilution), β-actin (ABclonal Technology, AC026, 1:20000 dilution), phospho-STAT1 (Sangon Biotechnology, D155017, 1:4500 dilution), anti-GFP (green fluorescent protein) (ABclonal Technology, AE078, 1:6000 dilution), horseradish peroxidase (HRP)-conjugated anti-Flag (AlpVHHS, 016–303-005, 1:5000 dilution), goat anti-rabbit (Invitrogen, 31460, 1:6000 dilution), and goat anti-mouse (Abcam, ab6789, 1:8000 dilution).

Techniques: Sequencing, Residue, Binding Assay

Journal: Veterinary Research

Article Title: Characterization of the rock pigeon type IV interferon system reveals conserved tyrosine-based signaling via the IFN-υR1/IL10RB complex

doi: 10.1186/s13567-026-01823-w

Figure Lengend Snippet: The rock pigeon IFN-υ activates the JAK-STAT signaling pathway and induces ISG expression . A Expression and detection of recombinant rock pigeon IFN-υ-Flag protein. HEK293T were transfected with a plasmid encoding rock pigeon IFN-υ-Flag. The cell culture supernatant was collected 48 h post-transfection. Samples of the supernatant were mixed with 5× SDS-PAGE loading buffer, denatured by heating at 100 °C for 10 min, and analyzed by Western blot using an anti-Flag antibody to detect the recombinant IFN-υ-Flag protein. B Rock pigeon IFN-υ activates the JAK-STAT signaling pathway. PEFs were stimulated with recombinant IFN-υ protein collected from transfected HEK293T (as in A ). Cells were harvested at 0, 18, 36, and 60 min post-stimulation. Cell lysates were prepared using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Activation of the pathway was assessed by Western blot analysis of STAT1 phosphorylation (p-STAT1). C – E Rock pigeon IFN-υ induces transcriptional upregulation of downstream ISGs. PEFs were stimulated with recombinant IFN-υ protein and harvested at 0, 3, 6, 12, and 24 h post-stimulation. Total RNA was extracted and reverse-transcribed into cDNA, and the mRNA expression levels of representative ISGs ( OAS1 , PKR , and RSAD2 ) were analyzed by qRT-PCR. Data are presented as mean ± SE. Statistical significance was determined by Student's t -test (* P < 0.05, ** P < 0.01, and ns for nonsignificant results).

Article Snippet: Commercial primary and secondary antibodies include Flag-Tag (Sigma-Aldrich, F1804, 1:6000 dilution), HA-Tag (Cell Signaling Technology, 3724S, 1:5000 dilution), STAT1 (ABclonal Technology, A19563, 1:5500 dilution), β-actin (ABclonal Technology, AC026, 1:20000 dilution), phospho-STAT1 (Sangon Biotechnology, D155017, 1:4500 dilution), anti-GFP (green fluorescent protein) (ABclonal Technology, AE078, 1:6000 dilution), horseradish peroxidase (HRP)-conjugated anti-Flag (AlpVHHS, 016–303-005, 1:5000 dilution), goat anti-rabbit (Invitrogen, 31460, 1:6000 dilution), and goat anti-mouse (Abcam, ab6789, 1:8000 dilution).

Techniques: Expressing, Recombinant, Transfection, Plasmid Preparation, Cell Culture, SDS Page, Western Blot, Lysis, Activation Assay, Phospho-proteomics, Reverse Transcription, Quantitative RT-PCR

Journal: Veterinary Research

Article Title: Characterization of the rock pigeon type IV interferon system reveals conserved tyrosine-based signaling via the IFN-υR1/IL10RB complex

doi: 10.1186/s13567-026-01823-w

Figure Lengend Snippet: Rock pigeon IFN-υ exerts antiviral functions through specific receptors IFN-υR1 and IL10RB . A – C IFN-υ induces ISG expression via IFN-υR1 and IL10RB, as determined by qRT-PCR of Mx ( A ), RSAD2 ( B ), and OASL ( C ) in DEFs with the indicated plasmid treatments and IFN-υ stimulation. D IFN-υ activates the JAK-STAT pathway via IFN-υR1 and IL10RB, as shown by Western blot of p-STAT1 in DEFs treated with indicated plasmids and IFN-υ. E , F Protein interaction between rock pigeon IFN-υR1 and duck STAT1 was assessed by Co-IP followed by Western blot. G , H IFN-υ inhibits DTMUV infection in DEFs via IFN-υR1 and IL10RB, as determined by qRT-PCR of DTMUV- E ( G ) and TCID₅₀ titration ( H ). I – L IFN-υ inhibits DTMUV infection in PEFs via IFN-υR1 and IL10RB, as determined by qRT-PCR of DTMUV- E and TCID 50 titration at 40 h post-infection (hpi) in cells either treated directly with IFN-υ ( I , J ) or transfected with the receptor combination prior to IFN-υ stimulation ( K , L ). Statistical analysis was performed using Student’s t -test (* P < 0.05, ** P < 0.01).

Article Snippet: Commercial primary and secondary antibodies include Flag-Tag (Sigma-Aldrich, F1804, 1:6000 dilution), HA-Tag (Cell Signaling Technology, 3724S, 1:5000 dilution), STAT1 (ABclonal Technology, A19563, 1:5500 dilution), β-actin (ABclonal Technology, AC026, 1:20000 dilution), phospho-STAT1 (Sangon Biotechnology, D155017, 1:4500 dilution), anti-GFP (green fluorescent protein) (ABclonal Technology, AE078, 1:6000 dilution), horseradish peroxidase (HRP)-conjugated anti-Flag (AlpVHHS, 016–303-005, 1:5000 dilution), goat anti-rabbit (Invitrogen, 31460, 1:6000 dilution), and goat anti-mouse (Abcam, ab6789, 1:8000 dilution).

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Infection, Titration, Transfection

Journal: Veterinary Research

Article Title: Characterization of the rock pigeon type IV interferon system reveals conserved tyrosine-based signaling via the IFN-υR1/IL10RB complex

doi: 10.1186/s13567-026-01823-w

Figure Lengend Snippet: Knockdown of rock pigeon IFN-υR1 or IL10RB attenuates IFN-υ-induced ISG expression. A , B Validation of knockdown efficiency for rock pigeon IFN-υR1 and IL10RB by qRT-PCR. C Knockdown of IFN-υR1 and IL10RB reduced IFN-υ-induced RSAD2 and STAT1 expression, as measured by qRT-PCR. D Overexpression of IFN-υR1 and IL10RB enhances IFN-υ-induced RSAD2 promoter activity. E Endogenous IFN-υR1 or IL10RB knockdown weakens the IFN-υ-mediated induction of the RSAD2 promoter. F , G Knockdown abolishes the potentiating effect of overexpressed IFN-υR1 or IL10RB on IFN-υ-induced RSAD2 promoter activity. Data are presented as mean ± SE from three independent experiments. * P < 0.05, ** P < 0.01 (Student’s t -test).

Article Snippet: Commercial primary and secondary antibodies include Flag-Tag (Sigma-Aldrich, F1804, 1:6000 dilution), HA-Tag (Cell Signaling Technology, 3724S, 1:5000 dilution), STAT1 (ABclonal Technology, A19563, 1:5500 dilution), β-actin (ABclonal Technology, AC026, 1:20000 dilution), phospho-STAT1 (Sangon Biotechnology, D155017, 1:4500 dilution), anti-GFP (green fluorescent protein) (ABclonal Technology, AE078, 1:6000 dilution), horseradish peroxidase (HRP)-conjugated anti-Flag (AlpVHHS, 016–303-005, 1:5000 dilution), goat anti-rabbit (Invitrogen, 31460, 1:6000 dilution), and goat anti-mouse (Abcam, ab6789, 1:8000 dilution).

Techniques: Knockdown, Expressing, Biomarker Discovery, Quantitative RT-PCR, Over Expression, Activity Assay

Journal: Veterinary Research

Article Title: Characterization of the rock pigeon type IV interferon system reveals conserved tyrosine-based signaling via the IFN-υR1/IL10RB complex

doi: 10.1186/s13567-026-01823-w

Figure Lengend Snippet: Functional identification of conserved tyrosine residues in the intracellular domain of rock pigeon IFN-υR1. A Two tyrosine residues are conserved in IFN-υR1 across vertebrates. In rock pigeon IFN-υR1, these correspond to Tyr 334 and Tyr 525 . B Schematic representation of wild-type (wt) rock pigeon IFN-υR1 and its tyrosine mutant constructs. C Tyr 334 and Tyr 525 are critical for STAT phosphorylation. PEFs were transfected with vector, IFN-υR1-Flag (wt), or its mutant plasmids. At 24 h post-transfection, cells were stimulated with IFN-υ for 1 h, then lysed and analyzed by Western blot for p-STAT1. D Tyr 334 and Tyr 525 are also essential for IFN-υ-induced ISG transcription. Similarly treated PEFs were harvested 24 h after IFN-υ stimulation. The mRNA expression levels of RSAD2 and STAT1 were measured by qRT-PCR. E , F Co-IP assays confirm the protein–protein interaction between rock pigeon IFN-υR1 and STAT1. G Tyr 334 and Tyr 525 of rock pigeon IFN-υR1 are also involved in its interaction with STAT1, as assessed by Western blot.

Article Snippet: Commercial primary and secondary antibodies include Flag-Tag (Sigma-Aldrich, F1804, 1:6000 dilution), HA-Tag (Cell Signaling Technology, 3724S, 1:5000 dilution), STAT1 (ABclonal Technology, A19563, 1:5500 dilution), β-actin (ABclonal Technology, AC026, 1:20000 dilution), phospho-STAT1 (Sangon Biotechnology, D155017, 1:4500 dilution), anti-GFP (green fluorescent protein) (ABclonal Technology, AE078, 1:6000 dilution), horseradish peroxidase (HRP)-conjugated anti-Flag (AlpVHHS, 016–303-005, 1:5000 dilution), goat anti-rabbit (Invitrogen, 31460, 1:6000 dilution), and goat anti-mouse (Abcam, ab6789, 1:8000 dilution).

Techniques: Functional Assay, Mutagenesis, Construct, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Blocking the m 6 Am methyltransferase PCIF1 releases STAT1-mediated Th1 immunity to potentiate cancer immunotherapy

doi: 10.1038/s41467-026-75269-1

Figure Lengend Snippet: a Schematic of m 6 Am-seq in naïve CD4 + T cells. b Metagene profiles of m 6 Am peak distribution along normalized transcripts. c KEGG pathway enrichment analysis of m 6 Am-modified genes in naïve CD4 + T cells. d Schematic of RiboTag-seq in naïve CD4 + T cells. e Volcano plots of differential translation efficiency (TE) between cKO and WT naïve CD4 + T cells based on RiboTag-seq data. TE was calculated as the ratio of Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) in RiboTag-seq to FPKM in input RNA-seq. f Venn diagram of TE-upregulated genes and m 6 Am-modified genes in naïve CD4 + T cells. g RiboTag-qPCR analysis of Stat1 mRNA association with ribosomes in naïve CD4 + T cells ( n = 4). h–j Representative immunoblot ( h ) and corresponding quantitative data ( i ) ( n = 8), along with representative histograms (left) and quantitative analysis (right) in ( j ) ( n = 6) reveal STAT1 expression in naïve CD4 + T cells from WT or cKO mice. k Schematic (left) and the quantification (right) of the dual-luciferase reporter assay used to characterize the effect of A-to-T substitution at the first nucleotide after the m 7 G cap in the 5’-UTR of Stat1 ( n = 12). l Quantification of IFN-γ expression in Th1-differentiated CD4 + T cells treated with a STAT1 inhibitor (WT or cKO Group n = 6, other Group n = 5). Each n represents biologically independent samples ( k ) or independent samples from individual mouse ( a , e , g – j , l ). Error bars represent means ± SEM; two-tailed, unpaired t -test ( g , i–k ); one-way ANOVA ( l ). p -values are shown on the graphs, and p -values < 0.0001 are reported as such.

Article Snippet: During the first 24 h of Th1 differentiation from naïve CD4 + T cells, the STAT1 inhibitor Fludarabine (2.5 μM; MedChemExpress, 21679-14-1) was added.

Techniques: Modification, RNA Sequencing, Western Blot, Expressing, Luciferase, Reporter Assay, Two Tailed Test