proteintech cat 66009 1 ig rrid ab 2687938  (Cell Signaling Technology Inc)

Journal: Journal of Neuroinflammation

Article Title: Phosphorylated TYK2 orchestrates the pathogenic program of CD4 + T cells in the development of CNS autoimmunity

doi: 10.1186/s12974-026-03934-9

Figure Lengend Snippet: TYK2 inhibition selectively modulates cytokine-induced STAT signaling in CD4 + T cells from naive mice. A , B Representative flow cytometry histograms and summary gMFI data showing the induction of p-TYK2 (unstimulated vs. stimulated) and the subsequent inhibition of p-STAT1 and p-STAT2 (vehicle vs. TYK2i) following stimulation with IFN-α A or IFN-β B in CD4 + T cells and in Th1 and Th17 subsets. C Levels of p-TYK2 induction and p-STAT3 inhibition following IL-23 stimulation in CD4 + T cells and in the Th1 and Th17 subsets. D Levels of p-TYK2 induction and p-STAT4 inhibition following IL-12 stimulation in CD4 + T cells and in the Th1 and Th17 subsets. E Levels of p-TYK2 induction and p-STAT5 inhibition following IL-2 stimulation in CD4 + T cells and Foxp3 + Treg. Cell populations are distinguished by color: CD4 + T cells (purple), Th1 subsets (red), Th17 subsets (blue), and Foxp3 + Tregs (red). Data are presented as mean ± SEM ( n = 5). Statistical significance was determined by unpaired two-tailed Student’s t-test (** p < 0.01, and *** p < 0.001, **** p < 0.0001, ns, not significant)

Article Snippet: In both mouse and human cells, phosphorylated signal transducer and activator of transcription(p-STAT) 1 (Tyr701), p‐STAT2 (Tyr690), and phosphorylated‐TYK2 (p-TYK2; Tyr1054/Tyr1055) were detected following stimulation with either IFN‐α (100 ng/mL; Abclonal, China) or IFN‐β (100 ng/mL; Abclonal).

Techniques: Inhibition, Flow Cytometry, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Phosphorylated TYK2 orchestrates the pathogenic program of CD4 + T cells in the development of CNS autoimmunity

doi: 10.1186/s12974-026-03934-9

Figure Lengend Snippet: TYK2 inhibition suppresses pathogenic cytokine production and selectively modulates STAT signaling in CD4 + T cells from AU patients. A Proportions of IFN-γ+, IL-17 A+, GM-CSF+, and Foxp3 + cells in patient-derived PBMCs stimulated with anti-CD3/CD28 for 3 days in the presence of vehicle or TYK2i. B , C Representative flow cytometry plots and summary gMFI data showing the induction of p-TYK2 (unstimulated vs. stimulated) and the subsequent inhibition of p-STAT1 and p-STAT2 (vehicle vs. TYK2i) following stimulation with IFN-α B or IFN-β C in CD4 + T cells. D Levels of p-TYK2 induction and p-STAT3 inhibition in CD4 + T cells following IL-23 stimulation. E Levels of p-TYK2 induction and p-STAT4 inhibition in CD4 + T cells following IL-12 stimulation. F Levels of p-TYK2 induction and p-STAT5 inhibition in CD4 + T cells following IL-2 stimulation. Data are mean ± SEM ( n = 5–8). Statistical significance was determined by unpaired two-tailed Student’s t-test (* p < 0.05, and *** p < 0.001, **** p < 0.0001, ns, not significant)

Article Snippet: In both mouse and human cells, phosphorylated signal transducer and activator of transcription(p-STAT) 1 (Tyr701), p‐STAT2 (Tyr690), and phosphorylated‐TYK2 (p-TYK2; Tyr1054/Tyr1055) were detected following stimulation with either IFN‐α (100 ng/mL; Abclonal, China) or IFN‐β (100 ng/mL; Abclonal).

Techniques: Inhibition, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Foci images of the DENV2 with ZIKV-equivalent STAT2-targeting mutations. B) Design of bimolecular fluorescence complementation (BiFC) assay for NS5-STAT2 interaction analysis in cells. C) Flow cytometry BiFC readouts of selected NS5 mutants with human STAT2. D) Foci images of DENV2 with single and combined STAT2-targeting mutations.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Bimolecular Fluorescence Complementation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Heatmap of contact analysis of NS5-STAT2 interacting residues from four available NS5-STAT2 complex structures (top and middle, each number represents the numbers of contacts at each residue) with the corresponding mutation fitness data derived from DMS of DENV2 in Vero cells (bottom) . B) Western-blot analysis of STAT2 degradation during infection by DENV2 with selected NS5 mutations at the STAT2-contacting residues. C) Multi-step replication kinetics of the DENV2-WT, -P336G, -N730D, -P336G+N730D, and - P336G+Q845A in Vero cells infected at MOI = 0.1. D) Relative binding of the NS5 mutants with human STAT2 by co-immunoprecipitation assay.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Residue, Mutagenesis, Derivative Assay, Western Blot, Infection, Binding Assay, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Foci images of DENV1-4 and ZIKV with P336G or N730D (DENV2 mutations). B) STAT2 degradation in A549 cells infected by DENV1-4 and ZIKV with P336G or N730D mutations. C) Multi-sequence alignment (MSA) of STAT2-interating residues across DENV1-4 (KDH, 16681, v1043, and H241, respectively) and ZIKV (SV0010) used in A) and B). MSA was performed with full-length NS5 sequences. The STAT2 interacting residues (based on the analysis performed in ) were then extracted from the aligned sequences and displayed using MView .

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Infection, Sequencing

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Type-I IFN sensitivity of DENV2 with MTase, STAT2-targeting, and combined mutations (MTase+STAT2) in A549-WT and A549-IFIT1-KO, STAT2-KO, and PAF1-KO. The A549 cells were treated with IFN-α2 at 500U/ml for 4 hours before virus infection at MOI = 0.1. The virus titers (FFU/ml) for calculating IFN sensitivity were measured at 2 dpi. B) Multi-step replication kinetics of the DENV2 panel in WT A549 (left plot) and IFIT1-KO A549 (right plot). The A549 cells were infected with DENV2 at MOI = 0.1. C) Western-blot analysis of STAT2 expression level from the multi-step replication kinetics experiment in B). D) Flow cytometry measurement of STAT2 levels (as mean fluorescence intensity, MFI) in the infected and the bystander WT- and -IFIT1-KO A549 at 1 and 2 dpi (left and middle plot) and in the WT- and KO cells treated with type-I IFN at 500U/ml (right plot). E) Proteomic analysis showing differential human protein levels between WT- and IFIT1KO A549 at 2 dpi infected with either DENV2-E217A or DENV2-S150T+P336G+N730D as in B) (left volcano plot). The right heatmap shows the expression profile of ISGs with similar profile to STAT2 in different A549 cells (x-axis). F) Measurement of secreted IFN-β and TNF-α (pg/ml) of A549-WT and -IFIT1-KO cells infected with one of the three viruses, DENV2-WT, - E217A, or S150T+P336G+N730D (MOI = 1) at 1 and 2 dpi. The data points in the top bar plot from each virus were aggregated by cell types. The bottom paired line plots were matched by virus, with the data points in the IFIT1-KO being an average of the measurements from three IFIT1-KO clones. Statistical significance is indicated by brackets above the data sets. Exact p value is provided for each comparison (* ; ns, not significant). Statistical analysis was performed using an unpaired Student’s t-test for the top bar plots and a paired t-test for the bottom line plots.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Virus, Infection, Western Blot, Expressing, Flow Cytometry, Fluorescence, Clone Assay, Comparison

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Multi-step replication kinetics of the mutant DENV2 in STAT2-KO and PAF1-KO A549 cells. A549 cells were infected with DENV2 at MOI = 0.1. B) Western-blot analysis of IFIT1 and dengue NS3 expression in wild-type A549 cells during multi-step replication kinetics analysis in A). C) Flow cytometry of DENV2 infection by anti-NS3 in WT and IFIT1-KO A549 cells. D) Volcano plots illustrating the distribution of quantified proteins. The x-axis represents the fold change (LFC), and the y-axis represents the statistical significance as (-value). Proteins with a fold change > 2 and a p-value < 0.01 were considered significantly differentially expressed. Selected ISGs upregulated during DENV infections in WT- and IFIT1-KO A549 are labeled by their gene symbols. Aggregate data of DENV2-E217A and DENV2-S150T+P336G+N730D were used to represent the data of infected cells. E-F) Comparison between the cytokine concentrations (pg/ml) secreted from WT-A549 and those from IFIT1-KO-A549 infected with one of the three viruses, DENV2-WT, -E217A, or S150T+P336G+N730D (MOI = 1) at 1 and 2 dpi. The data points in the top bar plots (E) from each virus were aggregated by cell types. The bottom paired line plots (F) were matched by virus, with the data points in the IFIT1-KO being an average of the measurement from three IFIT1-KO clones. Statistical significance is indicated by brackets above the data sets. Exact values are provided for each comparison (* ; ns, not significant). Statistical analysis was performed using an unpaired Student’s t-test for the top bar plots and a paired t-test for the bottom line plots. G) Dose-response analysis of IFN-α2 and STAT2 level in A549 cells. A549 cells were treated with IFN-α2 48 hours before measurement by flow cytometry.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Mutagenesis, Infection, Western Blot, Expressing, Flow Cytometry, Labeling, Comparison, Virus, Clone Assay

Journal: Cell Death & Disease

Article Title: USP24-dependent STAT2 stabilization mediates physiologic and pathologic bone formation

doi: 10.1038/s41419-026-08888-7

Figure Lengend Snippet: A C3H10T1/2 cells were transfected with either vector control or Flag- USP24 , and 48 h later, mRNA levels (left, n = 4) and protein levels (right) of USP24 were determined by qRT-PCR and immunoblotting, respectively. B Enrichment scores of putative USP24-binding proteins generated by WebGestalt pathway analysis. C Flag- USP24 and His-ubiquitin were transfected into HEK293T cells along with STAT1 , STAT2 , or STAT3 . After 48 h of transfection, cells were treated with 10 μM MG132 for 6 h, lysed, pulled down with Ni-NTA agarose, and immunoblotted with the indicated antibodies. D Flag- USP24 and HA-ubiquitin were transfected into C3H10T1/2 cells. 48 h later, cells were treated with 10 μM MG132 for 6 h, lysed, pulled down with Flag-conjugated agarose, and immunoblotted for STAT2. STAT2 protein levels ( E ) and mRNA levels ( F , n = 4) in shCtrl - or shUsp24 -expressing mouse COBs. G 10 11 GCs of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24 were injected into P0 neonates, and 28 days later, STAT2 protein levels in the tibia were assessed by immunoblotting. H shCtrl or shUsp24 -expressing mouse COBs were treated with 10 μM MG132 for 6 h and immunoblotted for STAT2. shCtrl or shStat2 -expressing mouse COBs were cultured under osteogenic conditions. Six days later, ALP activity ( I , n = 9) and osteoblast gene expression ( J , n = 4) were examined. Data are representative of three ( A, C–J ). A two-tailed unpaired Student’s t test for comparing two groups ( A, F, I, J ; error bars, SD of biological replicates).

Article Snippet: After sections were incubated with antibodies specific to USP24 (1:100, Novus Biologicals, NBP1-82942) or STAT2 (1:100, Abclonal, A14995) for 40 min at 37 °C, and secondary antibodies for 20 min at 37 °C.

Techniques: Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Binding Assay, Generated, Ubiquitin Proteomics, Expressing, Injection, Cell Culture, Activity Assay, Gene Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: USP24-dependent STAT2 stabilization mediates physiologic and pathologic bone formation

doi: 10.1038/s41419-026-08888-7

Figure Lengend Snippet: A H&E-stained sections (left) and IHC (middle and right) for USP24 in human tissue with heterotopic ossification (HO). Scale bar, 50 μm (left and middle) or 20 μm (right). B 8-week-old Acvr1 (R206H)Fl ; Cre-ER T2 mice were treated with intraperitoneal (i.p.) injection of tamoxifen for 5 days, and 3 days later, the pinch injury/cardiotoxin (CTX) injection was introduced to the gastrocnemius muscle. 24 h after the injury, the tibial muscle was dissected, and Usp24 mRNA levels were assessed by qRT-PCR ( n = 4). C Schematic diagram showing experimental strategy and plan. D 8-week-old Acvr1 (R206H)Fl ;Cre-ER T2 mice were treated with i.p. injection of tamoxifen for 5 days and 5 days later, 5 × 10 12 vg/kg of rAAV9.egfp was injected into the gastrocnemius muscle (i.m.), followed by CTX injection and pinch injury. 28 days later, EGFP expression was assessed by fluorescence microscopy. Scale bar, 100 μm. HO, heterotopic ossification; BM, bone marrow; M, muscle. E 5 × 10 12 vg/kg of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24 was injected into 8-week-old Acvr1 (R206H)Fl ;Cre-ER T2 mice as described in Fig. 4C, and Usp24 mRNA levels were assessed by qRT-PCR ( n = 6 for male and n = 5 for female). F Incidence (%) of HO-positive mice. The number of HO-positive mice is shown as a proportion of the total mice in each group ( n = 12 per group). In males, the incidence was 10/12 (83.3%) for the amiR-Ctrl- and 6/12 (50.0%) for the amiR-Usp24 -treated group ( p = 0.193). In females, 11/12 (91.7%) for the amiR-Ctrl- and 6/12 (50.0%) for the amiR - Usp24 -treated group ( p = 0.068). HO was detected by radiography and quantified by micro-CT. Representative radiographic images ( G ), quantified HO bone volume (BV, H , n = 5), and 3D reconstruction images ( I ) were shown. Scale bar, 1 mm ( I ). HO was assessed by H&E staining ( J ) and IHC for STAT2 ( K ) in rAAV-treated femurs. Scale bar, 250 μm (J, upper), 100 μm (J, lower), or 20 μm ( K ). IgG was used as a negative control. Data are representative of three ( A, D, G, I–K ). A two-tailed unpaired Student’s t test for comparing two groups ( B, E, H ; error bars, SD of biological replicates). For Fig. 4F, statistical significance was determined by Fisher’s exact test.

Article Snippet: After sections were incubated with antibodies specific to USP24 (1:100, Novus Biologicals, NBP1-82942) or STAT2 (1:100, Abclonal, A14995) for 40 min at 37 °C, and secondary antibodies for 20 min at 37 °C.

Techniques: Staining, Injection, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Micro-CT, Negative Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: USP24-dependent STAT2 stabilization mediates physiologic and pathologic bone formation

doi: 10.1038/s41419-026-08888-7

Figure Lengend Snippet: A H&E-staining (left) and IHC for STAT2 (right) in human HO tissue. Blue arrows indicate bone-lining cells; Orange arrows indicate bone-embedded osteocytes. Scale bar, 20 μm. B 8-week-old Acvr1 (R206H)Fl ; Cre-ER T2 mice were treated with i.p. injection of tamoxifen for 5 days, and 5 days later, the pinch injury and CTX injection were introduced to the gastrocnemius muscle. 24 h after injury, the tibial muscle was dissected, and Stat2 and Ifi204 mRNA levels were assessed by qRT-PCR ( n = 4). C Schematic diagram illustrating the mechanisms by which USP24 promotes osteoblast differentiation through STAT2 stabilization. Prrx1 + osteogenic progenitors isolated from 4-week-old Prrx1-Cre ( Acvr1 WT ) or Acvr1 (R206H)Fl ;Prrx1-Cre femurs, transduced with 5 × 10 10 GCs of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24 , and cultured in osteogenic induction medium (OIM) with or without activin A (50 ng/ml). ALP activity ( D , n = 6) and mRNA levels of osteoblast genes ( E , n = 4) were examined at day 6. Protein levels of USP24, phospho-SMAD1/5, and STAT2 were determined by immunoblotting at day 1 of osteogenic differentiation ( F ). Prrx1 + osteogenic progenitors isolated from 4-week-old Prrx1-Cre ( Acvr1 WT ) or Acvr1 (R206H)Fl ;Prrx1-Cre femurs were transduced with 5 × 10 10 GCs of rAAV9.egfp carrying amiR-Ctrl or amiR-Stat2 and cultured in OIM with or without activin A (50 ng/ml). ALP activity ( G , n = 6) and mRNA levels of osteoblast genes ( H , n = 4) were examined at day 6. I 5 × 10 12 vg/kg of rAAV9.egfp carrying amiR-Ctrl or amiR-Stat2 was injected into 8-week-old Acvr1 (R206H)Fl ;Cre-ER T2 mice as described in Fig. , and Stat2 mRNA levels were assessed by qRT-PCR ( n = 4). HO formation was assessed by micro-CT. 3D reconstruction images ( J ) and quantified HO bone volume (BV, K n = 8 for amiR-Ctrl and n = 6 for amiR-Stat2 ) were shown. Scale bar, 1 mm ( J ). Data are representative of three ( A, F, J ). A two-tailed unpaired Student’s t test for comparing two groups ( B, D, E, G–I, K ; error bars, SD of biological replicates).

Article Snippet: After sections were incubated with antibodies specific to USP24 (1:100, Novus Biologicals, NBP1-82942) or STAT2 (1:100, Abclonal, A14995) for 40 min at 37 °C, and secondary antibodies for 20 min at 37 °C.

Techniques: Staining, Injection, Quantitative RT-PCR, Isolation, Transduction, Cell Culture, Activity Assay, Western Blot, Micro-CT, Two Tailed Test