Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: The role of interleukin-11 in pregnancy involves up-regulation of alpha2-macroglobulin gene through janus kinase 2-signal transducer and activator of transcription 3 pathway in the decidua.

doi: 10.1210/me.2006-0296

Figure Lengend Snippet: Fig. 9. Jak2 But Not Jak1 or Tyk2 Is Phosphorylated by IL-11 in UIII Cells A, UIII cells were kept in serum free medium for 24 h before they were treated with IL-11 (100 ng/ml) for different lengths of time. Phosphorylation of Tyk2, Jak1, and Jak2 was studied by Western analysis. B, Total RNA obtained from UIII cells treated with different doses of AG490 and for different times was subjected to RT-PCR analysis, as described in Materials and Methods. The autoradiograms are representative of three separate experiments.

Article Snippet: Antibodies to phospho-Jak2, phospho-Tyk2 (Cell Signaling, Danvers, MA), Jak2, phopho-Stat5a/b, phospho-Stat3, Stat3 (Upstate Biotechnology, Lake Placid, NY), Stat5a/b, Jak1, phospho-Jak1 (BioSource International, Inc., Camarillo, CA), and Tyk2 (BD Biosciences, San Jose, CA) were used.

Techniques: Phospho-proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 1. TYK2 activity is not required for IFN-induced recruitment of PKD2 to IFNAR1. A, phosphorylation and levels of endogenous IFNAR1 immunopurified (IP) from 11.1-derived cells that harbor wild-type (WT-5) or catalytically inactive (KR-2) TYK2 cells treated with IFN as indicated. B, immu- noblot analysis of IFNAR1 immunoprecipitated from 293T cells treated with IFN for the indicated times. Left lane of the upper panel represents the whole celllysatefromuntreated293Tcells.C,immunoblotanalysisofIFNAR1immu- nopurified from WT-5 and KR-2 cells treated with IFN for the indicated times. Whole cell lysates (WCL) were used for detection of tyrosine phosphorylation of STAT1 and total STAT1.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: Activity Assay, Phospho-proteomics, Derivative Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 2. IFN stimulates kinase activity of PKD2 and its interaction with TYK2. A, activity of GST-PKD2 expressed in cells harboring wild-type or kinase-dead TYK2, and left untreated or treated with IFN, was analyzed by in vitro Ser-535 phosphorylation of GST-IFNAR1 as assessed by immunoblotting using a Ser(P)-535-specific antibody. Levels of substrate and kinase were also analyzed by immunoblotting. B, interaction between endogenous TYK2 and PKD2 in HeLa cells treated as indicated was analyzed by co-immunoprecipitation (IP) and immunoblotting. WCL, whole cell lysate. IgG, isotype antibody control. C, localization of endogenous TYK2 in 2fTGH cells or isogenic 11.1 cells was detected using primary anti-TYK2 antibody (1o Ab, where indicated) and secondary antibodies conjugated with Alexa Fluor 488. Nuclei were counterstained using DAPI. D, localization of endogenous PKD2 in 2fTGH cells transduced with the indicated shRNA was detected using primary anti-PDK2 antibody (1o Ab, where indicated) and secondary antibodies conjugated with Alexa Fluor 594. Nuclei were counterstained using DAPI. E, co-localization of TYK2 and PKD2 in 2fTGH cells treated with IFN (2000 units/ml for 5 min) or not was determined by confocal microscopy analysis using indicated antibodies.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Transduction, shRNA, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 3. TYK2 activity is required for IFN-induced tyrosine phospho- rylation of PKD2. A, phosphorylation and levels of endogenous PKD2 immu- nopurified (IP) from WT-5 or KR-2 cells treated with IFN as indicated were analyzed by the indicated antibodies. B, tyrosine phosphorylation and levels of endogenous PKD2 immunopurified from HeLa cells treated with IFN as indicated were analyzed by immunoblotting. C, HA-tagged TYK2 was expressed in and immunopurified from 293T cells that were IFN-treated (lanes1–3and5)ornot(lane4).TYK2proteinswerethenincubatedwithPKD2 purified from serum-starved and untreated 2fTGH cells (except in lane 2) in the presence of ATP (except in lane 2). The reaction was analyzed by immu- noblotting using an anti-phosphotyrosine antibody. The recombinant Src protein was used as positive control.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Purification, Recombinant, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 4. TYK2 is capable of phosphorylating PKD2 on Tyr-438. A, alignment of fragments of primary sequences of the plekstrin homology domains within human PDK1 (Prkd2) and PKD2 (Prkd2),; Tyr-463 and Tyr438 are underlined. B, in vitro tyrosine phosphorylation of purified from cells GST-PKD2 (wild-type or Y438F mutant, YF) by recombinant truncated GST-TYK2833–1187 kinase analyzed by immunoblotting (IB). C, phosphorylation and levels of GST-PKD2 (wild type orY438Fmutant)expressedin293TcellstreatedornotwithIFNasindicatedbeforeharvesting.GST-PKD2specieswerepurifiedfromthelysatesusingaffinity beads and analyzed by immunoblotting using the indicated antibodies. D, interaction of endogenous IFNAR1 with exogenous GST-tagged PKD2 (wild type or Y438Fmutant)expressedin293Tcellsdeterminedbyimmunoprecipitation(IP)followedbyimmunoblottingusingtheindicatedantibodies.E,specificTyr-438 phosphorylation of GST-tagged PKD2 species (wild type or Y438F mutant) purified from 293T cells treated or not with IFN for 5 min analyzed by immuno- blotting using the indicated antibodies. F, specific Tyr-438 phosphorylation of endogenous PKD2 immunopurified from HeLa cells treated with IFN for the indicated times analyzed by immunoblotting using the indicated antibodies.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: In Vitro, Phospho-proteomics, Purification, Mutagenesis, Recombinant, Western Blot

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: NanoSeq of TILs identified somatic mutations in TYK2 . a. A schematic of somatic genomic sequencing of patient derived TILs. b. Distribution of tumor patients types. c. Classification of TYK2 mutation types in TILs. d. Lollipop plots of TYK2 mutations in tumor-infiltrating CD4⁺ T cells and tumor-infiltrating CD8⁺ T cells.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Genomic Sequencing, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Lollipop plots of TYK2 mutations in TILs. a. Locations of TYK2 mutations in tumor-infiltrating B cells and tumor-infiltrating NK cells.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques:

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: TYK2 D810V expression enhances T cells effector function a. Western blot of Jurkat cells overexpressing 15 TYK2 missense mutations. Representative blot from two independent experiments. b. IFNγ and IL8 levels measured in primary T cells expressing empty vector, TYK2 WT , or TYK2 D810V with or without IL12 ( n= 3 replicates per group). c. Representative images of A549 tumor co-cultured with primary T cells overexpressed empty vector, TYK2 WT , or TYK2 D810V , scale bars = 1000 μm ( n= 3 replicates per group). d. Relative tumor area of A549 tumor co-cultured with primary T cells overexpressed empty vector, TYK2 WT , or TYK2 D810V ( n =3 replicates per group). e. IFNγ levels measured in A549 tumor cells co-cultured with primary T cells expressing empty vector, TYK2 WT , or TYK2 D810V ( n =3 replicates per group). f. Volcano plot showing upregulated genes in Jurkat T cells overexpressing TYK2 D810V compared to TYK2 WT ( n= 3 replicates per group). g. Enriched gene signatures associated with up- or down-regulated genes associated with TYK2 D810V compared to TYK2 WT . h. GSEA showing upregulation of Interferon-gamma response, Interferon-alpha response, Interferon-gamma response and IL6-JAK-STAT3 signaling genes in TYK2 D810V overexpressing Juakat T cells.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Expressing, Western Blot, Plasmid Preparation, Cell Culture

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Functional characterization of TYK2 D810V mutation and single-cell transcriptomic profiling of TYK2-deficient primary T cells. a . STAT3 reporter activity in TYK2-knockout 293T cells expressing empty vector, TYK2 WT or TYK2 D810V . b. A schematic of A549 tumor and T cell co-culture experiment. c. Volcano plot showing genes downregulated in TYK2-knockout primary human CD4⁺ T cells. d. Volcano plot showing genes downregulated in TYK2-knockout primary human CD8⁺ T cells. e. GSEA enrichment plot showing top enriched Hallmark gene sets ranked by NES in TYK2-knockout primary CD4⁺ T cells. f. GSEA enrichment plot showing top enriched Hallmark gene sets ranked by NES in TYK2-knockout primary CD8⁺ T cells.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Functional Assay, Mutagenesis, Activity Assay, Knock-Out, Expressing, Plasmid Preparation, Co-Culture Assay

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Design of Tyk2 D807V mutant mice. a. Genomic sequences for human TYK2 D810V and mouse Tyk2 D807V . b. Single-stranded DNA (ssDNA) and sgRNA used with CAS9 to generate the Tyk2 D807V mouse. c. Mouse TYK2 genomic sequences before and after CRISPR editing. d. Sanger sequencing for het Tyk2 D807V point mutation mice.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Mutagenesis, Genomic Sequencing, CRISPR, Sequencing

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: TYK2 D807V mice do not exhibit signs of autoimmunity or premalignancy. a. Representative appearance of 2- and 12-month-old het or homo Tyk2 D807V and Tyk2 WT mice. b. Body weights of the above mice. Males: WT ( n =8); het ( n =12); homo ( n =7). Females: WT ( n =6); het ( n =11); homo ( n =6). c. Serum levels of ALT and AST (U/L) of the above mice (WT ( n = 6); het ( n = 6); homo ( n = 6). mean ± s.e.m. is depicted. d. Representative H&E-stained liver sections from above mice (scale bars = 250 μm). e. Representative H&E-stained splenic sections from above mice (scale bars = 250 μm). f. Complete blood counts from 12-month-old mice. g. Representative spleens from above mice (scale bars = 10 mm). h. Representative western blot of liver tissues from 2-month old mice. For the data in this figure, each dot is one mouse. Error bars show mean ± s.e.m. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Tyk2 D807V mutant mice appear healthy with no obvious signs of disease. a. H&E of kidney sections of 2- and 12-month-old het or homo Tyk2 D807V mice and WT littermates (scale bars = 250 μm). b. Spleen-to-body weight ratios (n = 6 for each genotype). c. Complete blood counts of 2-month-old het or homo Tyk2 D807V mice and WT littermates (n = 6 for each genotype). For data in this figure, mean ± s.e.m. is depicted. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: TYK2 D807V mice show enhanced anti-tumor effects. a. Syngeneic MC38 growth in male Tyk2 WT , het, or homo Tyk2 D807V mice. The tumor number is shown as the “n” in the figure and two tumors per mouse were implanted. P values were determined by two-way ANOVA. b. Tumor weights from panel a . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. c. Representative tumors from each group in panel a were ranked by size in descending order and the top six are shown. d. Syngeneic MC38 growth in female het Tyk2 D807V mice and Tyk2 WT littermates. The tumor number is shown as the “n” in the figure and two tumors per mouse were implanted. P values were determined by two-way ANOVA. e. Quantification of tumor-infiltrating CD45⁺ cells analyzed by flow cytometry. Each data point indicates an individual tumor (WT ( n = 8); Het ( n = 8)). P values were determined using two-tailed unpaired t -tests. f. Quantification of CD4⁺ and CD8⁺ T cell subsets from tumors analyzed by flow cytometry. Each data point indicates an individual tumor (WT ( n = 8); Het ( n = 8)). P values were determined using two-tailed unpaired t -tests. g. Syngeneic MC38 growth +/- anti-PD-1 antibodies. The tumor number is shown as the “n” in the figure and two tumors per mouse were implanted. P values were determined by two-way ANOVA. h. Tumor weight collected from panel g . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. i. Representative tumors from each group in panel g were ranked by size in descending order and the top six are shown. j. Syngeneic RIL175 growth, +/- anti-PD-1 antibodies. The tumor number is shown as the “n” in the figure and two tumors per mouse were implanted. P values were determined by two-way ANOVA. k. Tumor weight collected from panel j . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. l. Representative tumors from each group in panel j were ranked by size in descending order and the top six are shown. m. Syngeneic B16 growth, +/- anti-PD-1 antibodies. The tumor number is shown as the “n” in the figure and two tumors per mouse were implanted. P values were determined by two-way ANOVA. n. Tumor weight collected from panel m . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. o. Representative tumors from each group in panel m were ranked by size in descending order and the top six are shown. p. Representative livers of Tyk2 D807V het and WT littermates with shp53/ NRAS G12V tumors, +/- anti-PD-1 antibodies (scale bar = 10 mm). q. Liver-to-body weight ratios from panel p . Each data point is one liver and P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. r. Representative livers of Tyk2 D807V het and WT littermates with CTNNB1 / MYC tumors, +/- anti-PD-1 antibodies (scale bar = 10 mm). s. Liver-to-body weight ratios from panel r . Each data point is one liver and P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For all data in this figure, error bars show mean ± s.e.m and statistical tests are stated above. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Tyk2 D807V mutant mice showed increased T cell infiltration upon tumor challenge. a. Representative flow cytometry gating strategy for immune cell subset identification from tumor-infiltrating CD45⁺ cells.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Mutagenesis, Flow Cytometry

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Tyk2 D807V reduces tumor burden in vivo through CD8⁺ T cells. a. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with anti-NK1.1 or anti-IgG antibody treatments. b. Tumor weights from panel a . c. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with anti-F4/80 or anti-IgG antibody treatments. d. Tumor weights from panel c . e. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with anti-Ly6G or anti-IgG antibody treatments. f. Tumor weights from panel e . g. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with anti-CD3 or anti-IgG antibody treatments. h. Tumor weights from panel g . i. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with anti-CD4 or anti-IgG antibody treatments. j. Tumor weights from panel i . k. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with anti-CD8 or anti-IgG antibody treatments. l. Tumor weights from panel k . m. Syngeneic MC38 growth in mice transplanted with CD8⁺ T cells from Tyk2 D807V het or WT littermates. n. Tumor weights from panel m . o. Representative tumors from panel m . p. Syngeneic RIL175 growth in mice transplanted with CD8⁺ T cells from Tyk2 D807V het or WT littermates. q. Tumor weights from panel p . r. Representative tumors from panel p . s. Syngeneic B16 growth in mice transplanted with CD8⁺ T cells from Tyk2 D807V het or WT littermates. t. Tumor weight collected from panel s . u. Representative tumors from panel s . All data in this figure are presented as mean ± s.e.m. Tumor cells were subcutaneously injected into both flanks of each mouse, so each mouse bears two tumors. Each data point represents an individual tumor (N). For syngeneic models growth curves (panels a,c,e,g,i,k,m,p,s ), P values were determined by two-way ANOVA. For tumor weight panels, P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test (panels b,d,f,h,j,i ), or two-tailed unpaired t -tests (panels n,q,t ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. Of note, the data in panels a - h share the same WT+anti-IgG and het+anti-IgG control groups. And the data in panels i-l share the same WT+anti-IgG and het+anti-IgG control groups. For all gross images, tumors from each group were ranked by size in descending order, and the top six are shown.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: In Vivo, Injection, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: The anti-tumor effects of TYK2 D807V mice are mediated by CD8⁺ T cells. a. MC38 syngeneic tumors from het Tyk2 D807V and WT littermates treated with anti-IgG, anti-CD3ε, anti-NK1.1, anti-F4/80 and anti-Ly6G antibodies. Tumors from each group were ranked by size in descending order, and the top six are shown. b. MC38 syngeneic tumors from het Tyk2 D807V and WT littermates treated with anti-IgG, anti-CD4 and anti-CD8β antibodies. Tumors from each group were ranked by size in descending order, and the top six are shown. c. Experimental schema for adoptive transplantation of CD8⁺ T cells.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Transplantation Assay

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Tyk2 D807V also impairs cancer growth in a cell-autonomous manner. a. Cell viability assay (CCK-8) performed on NRAS G12V / shp53 HCC cells isolated from WT and Tyk2 D807V het mice grown in culture ( n = 3,3). P values were determined by two-way ANOVA. a. b. NRAS G12V / shp53 HCC cells isolated from WT and Tyk2 D807V het mice were implanted into WT C57/B6 mice ( n = 26 Tyk2 WT and 22 Tyk2 D807V het tumors). b. Tumor weights from panel b . P values were determined using two-tailed unpaired t -test. c. Representative tumors from panel b . Tumors were ranked by size in descending order, and the top six are shown. d. Western blot of H2.35 cells overexpressing sg TYK2 knockout vectors. e. Transposon constructs containing sgRNA- NRAS G12V , shp53 , and a plasmid containing SB100 transposase. f. Representative livers of sg Tyk2 and sgCtrl with shp53/ NRAS G12V tumors (scale bar = 10 mm). g. Liver-to-body weight ratios from panel g . Each data point is one liver and P values were determined by two-tailed unpaired t -test. h. Surface tumor numbers from panel g . Each data point is one liver and P values were determined by two-tailed unpaired t -test. All data in this figure are presented as mean ± s.e.m. Tumor cells were subcutaneously injected into both flanks of each mouse, so each mouse bears two tumors. Each data point represents an individual tumor (N). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Viability Assay, CCK-8 Assay, Isolation, Two Tailed Test, Western Blot, Knock-Out, Construct, Plasmid Preparation, Injection

Journal: bioRxiv

Article Title: Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

doi: 10.1101/2025.10.13.682217

Figure Lengend Snippet: Pharmacological inhibition of TYK2 promotes tumor growth. a. Representative images of A549 tumor co-cultured with primary T cells after treatment with Deucravacitinib or vehicle, scale bars = 1000 μm ( n= 3 replicates per group). b. Relative tumor area of A549 tumor co-cultured with primary T cells after treatment with Deucravacitinib or vehicle ( n =3 replicates per group). c. IFNγ levels measured in A549 tumor cells co-cultured with primary T cells after treatment with Deucravacitinib or vehicle ( n =3 replicates per group). d. Syngeneic MC38 growth in mice receiving deucravacitinib ( n =16 tumors) or vehicle control ( n =14 tumors). e. Tumor weight collected from panel d . f. Representative IHC images of tumor infiltrating CD8⁺ cells in MC38 implanted tumors receiving deucravacitinib or vehicle. (scale bars = 150 μm) g. Quantification of tumor infiltrating CD8⁺ cell area of panel f ( n =10,10 for vehicle, deucravacitinib treated tumors). h. Syngeneic RIL175 tumor growth in mice receiving deucravacitinib ( n =14 tumors) or vehicle control ( n =16 tumors). i. Tumor weight collected from panel h . j. Representative syngeneic tumors from panel h . Tumors from each group were ranked by size in descending order, and the top six were selected for imaging. k. Syngeneic B16 tumor growth in mice receiving deucravacitinib ( n =12 tumors) or vehicle control ( n =12 tumors). l. Tumor weight collected from panel k . m. Representative syngeneic tumors from panel k . Tumors from each group were ranked by size in descending order, and the top six were selected for imaging. n. Syngeneic MC38 growth in Tyk2 D807V het or WT littermates, with vehicle or deucravacitinib treatments. o. Tumor weights from panel n . p. Representative syngeneic tumors from panel n . Tumors from each group were ranked by size in descending order, and the top six were selected for imaging. All data in this figure are presented as mean ± s.e.m. Tumor cells were subcutaneously injected into both flanks of each mouse, so each mouse bears two tumors. Each data point represents an individual tumor (N). For syngeneic models growth curves (panels d,h,k,n ), P values were determined by two-way ANOVA. For panels e,g,i,l,o , P values were determined by two-tailed unpaired t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TYK2 (Proteintech #16412-1-AP, 1:1000), TYK2 (Cell Signaling Technology #35615S, 1:1000), Phospho-TYK2 (Cell Signaling Technology, 68790S, 1:1000), STAT1 (Cell Signaling Technology, 14994S, 1:1000), Phospho-STAT1 (Cell Signaling Technology, 9167S, 1:1000), STAT3 (Cell Signaling Technology, 9139T, 1:1000), Phospho-STAT3 (Cell Signaling Technology, 9145S, 1:1000) and β-actin (Cell Signaling #4970, 1:1000).

Techniques: Inhibition, Cell Culture, Control, Imaging, Injection, Two Tailed Test

Journal: Clinical Cancer Research

Article Title: MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

doi: 10.1158/1078-0432.ccr-22-3722

Figure Lengend Snippet: Figure 1. TYK2 inhibitors reduce proliferation in MPNST cells. A, TYK2 protein levels were visualized by IHC in MPNST and plexiform neurofibroma, with positive staining scored on a 0 to 3 scale. B, Relative TYK2 protein levels in MPNST cell lines by WES Western blot analysis. Protein bands analyzed by densitometry, with TYK2 normalized to b-actin. C, JW23.3 and JH-2–002 cells were treated with TYK2 inhibitors (WU-12, WU-76) or inactive control (WU-18) for 3 days, and cell confluence was determined by IncuCyte assay. D, Representative images of IncuCyte assay at 72 hours, with YOYO-1 green fluorescence as indicator of apoptosis.

Article Snippet: Primary antibodies included total STAT3 (4904S; RRID:AB_331269), pSTAT3 (Tyr705; 9145S; RRID: AB_2491009), total p44/42 ERK1/2 (4695S; RRID:AB_390779), and pERK1/2 (Thr202/Tyr204; 4370S; RRID:AB_2315112), all from Cell Signaling Technologies, and TYK2 (A2128; Abclonal; RRID: AB_2764147).

Techniques: Staining, WES Western Blot, Control

Journal: Clinical Cancer Research

Article Title: MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

doi: 10.1158/1078-0432.ccr-22-3722

Figure Lengend Snippet: Figure 2. TYK2 inhibition leads to compensatory stimulation of the MAPK pathway in MPNST cells. A, JH-2–002 cells were treated with 40 mmol/L WU-12 or control for 48 hours, and gene expression was analyzed by qPCR array for JAK/STAT pathway–related genes. Log2 fold changes of significantly changed genes are graphed. B, JW23.3 cellswere treated with control or 40 mmol/LWU-12 or WU-76 for 48 hours. Global gene expressionwas determined byRNA-seq pathway analysis. Changes in gene expression for WU-12 versus control were examined by RNA-seq for genes downstream of STAT3 in a heatmap (C) and boxplot (D), as well as MEK/MAPK pathway genes in a volcano plot (E), enrichment plot (F), and boxplot (G). P < 0.05 vs. vehicle control.

Article Snippet: Primary antibodies included total STAT3 (4904S; RRID:AB_331269), pSTAT3 (Tyr705; 9145S; RRID: AB_2491009), total p44/42 ERK1/2 (4695S; RRID:AB_390779), and pERK1/2 (Thr202/Tyr204; 4370S; RRID:AB_2315112), all from Cell Signaling Technologies, and TYK2 (A2128; Abclonal; RRID: AB_2764147).

Techniques: Inhibition, Control, Gene Expression, RNA Sequencing

Journal: Clinical Cancer Research

Article Title: MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

doi: 10.1158/1078-0432.ccr-22-3722

Figure Lengend Snippet: Figure 3. TYK2 inhibitory drugs decrease acti- vation of STAT3 while increasing activation of ERK1/2 in MPNST cells. A, JW23.3 cells and (B) JH-2–002 cells were incubated with a TYK2 inhibitor (40 mmol/L WU-12) or vehicle control for the indicated times. Phosphorylat- ed and total protein levels for STAT3 and ERK1/2 were analyzed by the WES Western system. Bands were analyzed by densitometry in the WES software, with phosphorylated protein normal- ized to the matching total protein and expressed as percent of control at the same time point. , P < 0.05 vs. vehicle control at the same time point.

Article Snippet: Primary antibodies included total STAT3 (4904S; RRID:AB_331269), pSTAT3 (Tyr705; 9145S; RRID: AB_2491009), total p44/42 ERK1/2 (4695S; RRID:AB_390779), and pERK1/2 (Thr202/Tyr204; 4370S; RRID:AB_2315112), all from Cell Signaling Technologies, and TYK2 (A2128; Abclonal; RRID: AB_2764147).

Techniques: Activation Assay, Incubation, Control, Western Blot, Software

Journal: Clinical Cancer Research

Article Title: MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

doi: 10.1158/1078-0432.ccr-22-3722

Figure Lengend Snippet: Figure 4. The specific TYK2 inhibitor deucravacitinib (BMS-986165) decreases MPNST cell proliferation at lower doses.The MPNST celllines JW23.3 (A and B), JH-2–002 (C and D), and MPNST-724 (E and F) were treated with the indicated doses of the specific TYK2 inhibitor deucravacitinib or the pan-JAK inhibitor baricitinib for 3 days in IncuCyte live cell proliferation assays.

Article Snippet: Primary antibodies included total STAT3 (4904S; RRID:AB_331269), pSTAT3 (Tyr705; 9145S; RRID: AB_2491009), total p44/42 ERK1/2 (4695S; RRID:AB_390779), and pERK1/2 (Thr202/Tyr204; 4370S; RRID:AB_2315112), all from Cell Signaling Technologies, and TYK2 (A2128; Abclonal; RRID: AB_2764147).

Techniques:

Journal: Clinical Cancer Research

Article Title: MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

doi: 10.1158/1078-0432.ccr-22-3722

Figure Lengend Snippet: Figure 5. Inhibitors of TYK2 (deucravacitinib) and MEK (mirdametinib) act synergistically to reduce proliferation and increase apoptosis in JW23.3 MPNST cells. Cell confluence and apoptosis were analyzed by the IncuCyte assay after 48-hour incubation with drugs. Synergy was analyzed using Synergy Finder software by the HSA method for (A) inhibition of cell proliferation or (B) apoptosis, and mean synergy score is reported (P < 0.05 vs. drug alone). Intensity of red color indicates synergy score for each dose combination, whereas green indicates antagonism. C, Representative images of JW23.3 cells treated for 48 hours. YOYO-1 green fluorescence indicates apoptotic cells.

Article Snippet: Primary antibodies included total STAT3 (4904S; RRID:AB_331269), pSTAT3 (Tyr705; 9145S; RRID: AB_2491009), total p44/42 ERK1/2 (4695S; RRID:AB_390779), and pERK1/2 (Thr202/Tyr204; 4370S; RRID:AB_2315112), all from Cell Signaling Technologies, and TYK2 (A2128; Abclonal; RRID: AB_2764147).

Techniques: Incubation, Software, Inhibition

Journal: Clinical Cancer Research

Article Title: MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

doi: 10.1158/1078-0432.ccr-22-3722

Figure Lengend Snippet: Figure 6. The combination of drugs inhibiting TYK2 and MEK block MPNST tumor growth in mice. A, Schematic diagram of treatment paradigm. Mice with JW23.3 MPNST xenograft tumors (n ¼ 6 per group; B), WU-386 MPNST PDX tumors (n ¼ 3 per group; C), or JH-2–002 MPNST xenograft tumors (n ¼ 5 per group; D) were treated daily with 1.5 mg/kg mirdametinib (Mirda), 30 mg/kg deucravacitinib (Deucra, BMS-986165), the combination of drugs, or vehicle control for 3 weeks or until tumors reached the maximum allowed volume. , P < 0.05 vs. vehicle control; a, P < 0.05 for drug combination vs. drugs alone. E and F, Diagram of TYK2/STAT3 and MEK/ERK pathways after treatment with Deucra and/or Mirda in MPNST cells. (Illustrations were created with BioRender.com.)

Article Snippet: Primary antibodies included total STAT3 (4904S; RRID:AB_331269), pSTAT3 (Tyr705; 9145S; RRID: AB_2491009), total p44/42 ERK1/2 (4695S; RRID:AB_390779), and pERK1/2 (Thr202/Tyr204; 4370S; RRID:AB_2315112), all from Cell Signaling Technologies, and TYK2 (A2128; Abclonal; RRID: AB_2764147).

Techniques: Blocking Assay, Control

Journal: bioRxiv

Article Title: A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans

doi: 10.1101/2024.05.23.595417

Figure Lengend Snippet: a, Schematic diagram of the genome-wide CRISPR screen used to identify genes required for susceptibility to candidalysin-induced epithelial cell damage and scatterplot of the results. In the scatterplot, the number of sgRNAs targeting each gene that were significantly enriched is indicated with different symbols. The sigmaFC gene rank for each gene was calculated by adding the fold changes of all sgRNAs that target that gene, multiplied by the number of sgRNAs that showed significant enrichment. The top 7 enriched genes (sigmaFC > 6) are labeled in the plot. b, Effects of CRISPR deletion ( B3GAT3, XYLT2, B3GALT6, SLC39A9, EMP1 ), pharmacologic inhibition (Tyk2), and siRNA knockdown ( GBF1 ) on the survival of oral epithelial cells after 6 h of candidalysin exposure. The left panels show the survival (measured by an XTT assay) of epithelial cells exposed to the indicated concentrations of candidalysin. The plots represent the combined results of 3-4 experiments, each performed in triplicate. The right panels show the concentration of candidalysin that yielded 50% survival (IC 50 ), which was calculated from the data in the corresponding graph in the left panels. Results are mean ± SD. K/O, knockout; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 by the unpaired, two-sided Student’s t test.

Article Snippet: Twenty nanomolar GBF1 siRNA (Santa Cruz Biotechnology, #SC-105388), tyk2 siRNA (Santa Cruz Biotechnology, #SC-36764) or control scramble siRNA (Santa Cruz Biotechnology, #SC-37007) was applied to TR146 cell twice (time 0 h and time 24 h).

Techniques: Genome Wide, CRISPR, Labeling, Inhibition, Knockdown, XTT Assay, Concentration Assay, Knock-Out

Journal: bioRxiv

Article Title: A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans

doi: 10.1101/2024.05.23.595417

Figure Lengend Snippet: a, Western blotting of B3gat3, Xylt2, B3galt6, Slc39a9 and Gbf1 in the corresponding CRISPR knockout cells or siRNA knockdown cells. Arrows indicate the target proteins. b, Tyk2 siRNA knockdown does not affect survival of oral epithelial cells after 6-h of candidalysin exposure. The left panel shows a representative Tyk2 western blot, and the middle panel shows the survival (measured by an XTT assay) of epithelial cells exposed to the indicated concentrations of candidalysin. The plots represent the combined results of 3 experiments, each performed in triplicate. The right panels show the concentration of candidalysin that yielded 50% survival (IC 50 ), which was calculated from the data in the corresponding graph in the middle panel. Results are mean ± SD. ns, not significant by the unpaired, two-sided Student’s t test. c , Representative immunofluorescence images of TR146, B3GAT3 -/- , XYLT2 -/- and B3GALT6 -/- cells stained with an anti-heparan sulfate antibody (red) and DAPI (blue). Scale bar: 50μm. d , Flow cytometric analysis of heparan sulfate expression on the surface of TR146 cells and the indicated mutants.

Article Snippet: Twenty nanomolar GBF1 siRNA (Santa Cruz Biotechnology, #SC-105388), tyk2 siRNA (Santa Cruz Biotechnology, #SC-36764) or control scramble siRNA (Santa Cruz Biotechnology, #SC-37007) was applied to TR146 cell twice (time 0 h and time 24 h).

Techniques: Western Blot, CRISPR, Knock-Out, Knockdown, XTT Assay, Concentration Assay, Immunofluorescence, Staining, Expressing

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) Immunoblot assessment of levels of active, autophosphorylated pTYK2 and pJAK1, reflecting activities of the respective Janus kinases, as a function of vIRF-1 expression. Cultures of 293T cells were transfected with different amounts of the kinase vectors (0.15–0.60 μg) along with vIRF-1 expression plasmid; one dose (*, 0.5 μg) of TYK2 or JAK1 vector was transfected with RFP-expressing plasmid (negative control). Relative levels of TYK2, JAK1, and vIRF-1 expression along with β-actin (loading control) were assessed by immunoblotting. (B) Luciferase reporter (4xM67-Luc)-based analysis of functional suppression of TYK2 activity by vIRF-1. 293T cells were cotransfected with the STAT3-responsive reporter and different amounts of TYK2 expression plasmid, along with either empty vector (vector) or vIRF-1 expression plasmid. Luciferase activities in cell extracts are shown relative to levels (set at 1) for empty-vector-transfected cells lacking TYK2 overexpression (-). Average values from triplicate samples along with standard deviations are shown. The blots below the chart verify appropriate, vector-dose-dependent expression of TYK2 (in combined triplicate lysates). (C) Analysis of potential vIRF-1 effects on TYK2-STAT3 and TYK2-gp130 interactions in response to vIL-6 stimulation. Transfected-cell lysates containing TYK2-S in the absence (vec) or presence of vIRF-1 (v1) were subjected to S-protein-based AP and precipitated material was immunoblotted for detection of coprecipitated gp130 and total and phosphorylated STAT3. Tyrosine-phosphorylated (active) TYK2 (pTyk2) was also detected, along with vIRF-1. (D) Interaction of bacterially-produced and purified GST-tagged vIRF-1 (v1-GST) and Flag-tagged TYK2. AP of GST-vIRF-1 or GST (control) with glutathione beads and Flag-immunoblotting for coprecipitated TYK2 identified specific and direct interaction of vIRF-1 and TYK2. The asterisk indicates degradation products.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Control, Luciferase, Functional Assay, Activity Assay, Over Expression, Produced, Purification

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) HHV-8 (BAC16) infected iSLK cells were depleted of either TYK2 or JAK1 via lentiviral vector-mediated shRNA expression, using two different shRNAs for each target; non-silencing (ns) shRNA was used as a control. The cultures were left untreated (latent) or treated with Dox/NaB to induce lytic replication, and cells were harvested after six days for immunoblot analysis of pSTAT3, total-STAT3, and lytically expressed vIRF-1 and vIL-6. Relative levels of pSTAT3, normalized to total-STAT3, are shown below the pSTAT3 blots. (B) Analysis of pSTAT3 and pTYK2 levels as a function of vIRF-1 expression in iSLK cells, infected with native (WT) and vIRF-1-knockout (v1 ttg ) BAC16 viruses. Cultures were ether untreated (latent) or treated with Dox/NaB (lytic) for six days. Immunoblotting of whole-cell lysates was carried out to detect levels of pSTAT3 and pTYK2 relative to total STAT3 and TYK2 and lytic markers vIRF-1 and vIL-6. Charts show quantified data from triplicate lytic cultures, with WT values set at 1. Student’s t-test P values (two-tailed) are shown.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Infection, Plasmid Preparation, shRNA, Expressing, Control, Western Blot, Knock-Out, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) Verification of screening-identified vIRF-1Δ9 (deleted of residues 198–222) as defective for TYK2 interaction. This variant, along with STAT3-binding-defective Δ2 and Δ3 , were compared for TYK2 and STAT3 interactions by immunoprecipitation (IP) assay employing vIRF-1-Flag as “bait” and immunoblotting for vector-expressed S-tagged TYK2 or endogenous STAT3 in the precipitates. (B) Reciprocal precipitation for vIRF-1-TYK2 interaction, using affinity precipitation (AP) of TYK2-CBD and detection of coexpressed vIRF-1 or vIRF-1Δ9. (C) Testing of vIRF-1Δ9 for suppression of vIL-6-induced STAT3 signaling by transfection-based 4xM67-Luc reporter assay. The variant was inactive in this assay but retained inhibitory effect on the Smad-responsive 4xSBE-Luc reporter, used in parallel transfections. Immunoblotting for Flag-tagged vIRF-1, Smad3, and vIL-6 confirmed appropriate expression of the respective proteins in transfected-cell lysates (combined triplicates). (D) Diminished functional interaction of vIRF-1Δ9 with TYK2 was confirmed by analyzing cell lysates for pTYK2 and pSTAT3 levels in lentiviral vector-transduced, TYK2-overexpressing 292T cells (293T-TYK2) transfected with vIRF-1, vIRF-1Δ9, or RFP (negative control) expression plasmids. Levels of pTYK2 and pSTAT3, normalized to TYK2 and STAT3, respectively, and expressed relative to levels (set at 1) in the RFP vector-transfected culture are shown below the respective blots.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Variant Assay, Binding Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Affinity Precipitation, Transfection, Reporter Assay, Expressing, Functional Assay, Negative Control

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) Restriction profiling, using SpeI endonuclease, of wild-type (WT), vIRF-1Δ9-expressing (Δ9), and Δ9-mutation-repaired (Δ9 R ) HHV-8 BAC16 genomes. The white and grey arrowheads indicate the positions of DNA fragments containing WT/repaired and Δ9 vIRF-1 ORFs, respectively. (B) LANA-immunoblot verification of equivalent latent viral loads in iSLK cultures infected with the same infectious doses of native, vIRF-1 Δ9-mutated, and repaired (control) viruses. (C) Assessment of active STAT3 in recombinant HHV-8-infected and lytically reactivated iSLK cultures using the STAT3-responsive 4xM67-Luc reporter vector, transfected into the respective cultures. Luciferase activities from triplicate cultures are expressed as means, with standard deviations indicated; the mean value for WT virus is set at 1. Significance ( P ) of the increased luciferase activity in the vIRF-1Δ9-expressing cells was determined by student’s t-test. (D) Immunoblot analysis of pSTAT3 levels in lytically reactivated iSLK cells infected with WT, Δ9, and Δ9 R viruses. Cells were harvested at the indicated times after Dox/NaB treatment and lysates probed for total- and phospho-STAT3, in addition to LANA and lytically-induced vIRF-1 and vIL-6. Quantified levels of pSTAT3, normalized to total-STAT3 and relative to “WT” pSTAT3 levels (set at 1), are shown below the pSTAT3 blots. (E) Combined “day-2” pSTAT3/STAT3 data from panel D and two additional experiments. Average values relative to wild-type (set at 1), standard deviations, and P value (student’s t-test, two-tailed) for Δ9 against wild-type virus are shown. (F) Analysis of pSTAT3 and pTYK2 levels in lysates of lytically reactivated iSLK/BAC16 cultures, incorporating vIL-6-null (v6 ttg ) and vIL-6-null/vIRF-1Δ9 (v6 ttg +Δ9) viruses, harvested 2 days after Dox/NaB treatment. Levels of pSTAT3 and pTYK2, normalized to total-STAT3 and -TYK2 and expressed relative to levels (set at 1) in WT BAC16-infected cells, are indicated below the respective blots. (G) A similar analysis of pSTAT3 levels was undertaken using iTIME cells, harvested 2 days after lytic induction.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Expressing, Mutagenesis, Western Blot, Infection, Control, Recombinant, Plasmid Preparation, Transfection, Luciferase, Virus, Activity Assay, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: Data presented here have revealed direct interaction of vIRF-1 with STAT3 (phosphorylated and unphosphorylated) and its kinase TYK2. Abrogation of TYK2 interaction by the Δ9 mutation in vIRF-1 led to loss of suppression by vIRF-1 of active, phosphorylated STAT3 (pSTAT3) whereas STAT3 targeting, disrupted by vIRF-1 Δ2 and Δ3 mutations, had no effect. However, the latter interaction correlated with normal expression of vIRF-1, which was reduced in infected cells by the Δ2 and Δ3 mutations (effecting greatly diminished virus replication) and enhanced by peptide 1–72 (pep.1-72) that promotes vIRF-1-STAT3 interaction. Also, STAT3 depletion in infected cells led to reduced expression of vIRF-1 during lytic replication (not indicated). While the Δ9 mutation diminished virus productive replication in recombinant virus-infected cells, pep.1-72 promoted virus production. Levels of pSTAT3 in recombinant vIRF-1Δ9-virus-infected cells supporting lytic reactivation were higher than in cells infected with wild-type virus or vIRF-1Δ2 or vIRF-1Δ3 viruses. Introduction of the hyper-active, dephosphorylation-resistant STAT3C variant into infected cells inhibited productive replication, indicating that excessive pSTAT3 is detrimental to virus replication. Overall, the presented data show that vIRF-1 can suppress levels of active STAT3 via interaction with and inhibition of TYK2, likely helping to achieve levels of pSTAT3 compatible with efficient virus replication, while vIRF-1 interaction with STAT3 can effect positive regulation of vIRF-1, a viral protein known to support productive replication, and promote replication via this and/or other mechanisms.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Mutagenesis, Expressing, Infection, Virus, Recombinant, De-Phosphorylation Assay, Variant Assay, Inhibition