α 470 py stat4  (Cell Signaling Technology Inc)

Journal: JCI Insight

Article Title: Aiolos promotes CXCR3 expression on Th1 cells via positive regulation of IFN- γ /STAT1 signaling

doi: 10.1172/jci.insight.180287

Figure Lengend Snippet: ( A ) Publicly available chromatin immunoprecipitation sequencing (ChIP-Seq) data for STAT1 (GSM994528), STAT4 (GSM550303), and T-bet (GSM836124) were examined at Cxcr3 . Sequencing tracks were viewed using the Integrative Genomics Viewer (IGV). Regulatory regions of interest with transcription factor enrichment are indicated by the blue boxes. ( B ) Published RNA-Seq data (GSE203065) from in vitro–generated WT and Ikzf3 –/– Th1 cells were analyzed for DEGs. A heatmap of DEGs associated with IFN-γ/STAT1 and IL-12/STAT4 signaling in Th1 cells is shown, as well as additional genes involved in both pathways and Th cell differentiation. Gene names color-coded in blue are downregulated in Ikzf3 –/– Th1 cells. Note: Cxcr3 transcript data presented here are the same as in . ( C ) Schematic of proposed model in which Aiolos may regulate CXCR3 via impacts on components of the IFN-γ/STAT1 and IL-12/STAT4 cytokine signaling pathways. The downward arrows in blue indicate genes that are downregulated in the absence of Aiolos.

Article Snippet: The following antibodies were used to detect proteins: α-JAK2 (1:1,000; 3230, Cell Signaling Technology), α–pY-STAT4 (1:1,000; 5267, Cell Signaling Technology), α-STAT4 (1:1,000; 2653S, Cell Signaling Technology), α–pY-STAT1 (1:1,000; 9167S, Cell Signaling Technology), α-STAT1 (1:1,000; sc-417, Santa Cruz Biotechnology), α-Aiolos (1:20,000; 39293, Active Motif), α–β-actin–HRP (1:15,000; A00730, GenScript), goat α-mouse (1:5,000; 115-035-174, Jackson Immunoresearch), and mouse α-rabbit (1:5,000–1:10,000; sc-2357, Santa Cruz Biotechnology).

Techniques: ChIP-sequencing, Sequencing, RNA Sequencing, In Vitro, Generated, Cell Differentiation, Protein-Protein interactions

Journal: JCI Insight

Article Title: Aiolos promotes CXCR3 expression on Th1 cells via positive regulation of IFN- γ /STAT1 signaling

doi: 10.1172/jci.insight.180287

Figure Lengend Snippet: ( A ) Publicly available chromatin immunoprecipitation sequencing (ChIP-Seq) data for STAT1 (GSM994528), STAT4 (GSM550303), and T-bet (GSM836124) were examined at Cxcr3 . Sequencing tracks were viewed using the Integrative Genomics Viewer (IGV). Regulatory regions of interest with transcription factor enrichment are indicated by the blue boxes. ( B ) Published RNA-Seq data (GSE203065) from in vitro–generated WT and Ikzf3 –/– Th1 cells were analyzed for DEGs. A heatmap of DEGs associated with IFN-γ/STAT1 and IL-12/STAT4 signaling in Th1 cells is shown, as well as additional genes involved in both pathways and Th cell differentiation. Gene names color-coded in blue are downregulated in Ikzf3 –/– Th1 cells. Note: Cxcr3 transcript data presented here are the same as in . ( C ) Schematic of proposed model in which Aiolos may regulate CXCR3 via impacts on components of the IFN-γ/STAT1 and IL-12/STAT4 cytokine signaling pathways. The downward arrows in blue indicate genes that are downregulated in the absence of Aiolos.

Article Snippet: The following antibodies were used to detect proteins: α-JAK2 (1:1,000; 3230, Cell Signaling Technology), α–pY-STAT4 (1:1,000; 5267, Cell Signaling Technology), α-STAT4 (1:1,000; 2653S, Cell Signaling Technology), α–pY-STAT1 (1:1,000; 9167S, Cell Signaling Technology), α-STAT1 (1:1,000; sc-417, Santa Cruz Biotechnology), α-Aiolos (1:20,000; 39293, Active Motif), α–β-actin–HRP (1:15,000; A00730, GenScript), goat α-mouse (1:5,000; 115-035-174, Jackson Immunoresearch), and mouse α-rabbit (1:5,000–1:10,000; sc-2357, Santa Cruz Biotechnology).

Techniques: ChIP-sequencing, Sequencing, RNA Sequencing, In Vitro, Generated, Cell Differentiation, Protein-Protein interactions

Journal: The Journal of Biological Chemistry

Article Title: Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation *

doi: 10.1074/jbc.M112.361709

Figure Lengend Snippet: Sequences of primers, probes, and siRNA oligonucleotides used F, forward; R, reverse; FAM, carboxyfluorescein ; TAMRA, tetramethylrhodamine.

Article Snippet: Proteins were detected using the following antibodies: mouse α-PIM1 (12H8, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit α-PIM2 (ATLAS Antibodies, Stockholm, Sweden or D1D2, Cell Signaling Technology, Beverly, MA), rabbit α-PIM3 (D17C9, Cell Signaling Technology), mouse α-T-BET (4B10, Santa Cruz Biotechnology), rabbit α-STAT1 p84/p91 (E-23, Santa Cruz Biotechnology), rabbit α-STAT4 (C-20, Santa Cruz Biotechnology), mouse α-STAT6 (BD Biosciences), mouse α-GAPDH (number 5G4, 6C5, HyTest, Turku, Finland), and mouse α-β-actin (Sigma-Aldrich).

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation *

doi: 10.1074/jbc.M112.361709

Figure Lengend Snippet: Expression of PIM kinases in Th cells is regulated by the knockdown of STAT4 and STAT6. A , expression of PIM1, PIM2, and PIM3 during human Th cell differentiation. Naïve CD4+ Thp cells isolated from cord blood were activated (Th0) or stimulated with IL-12 (Th1) or IL-4 (Th2). Samples were harvested for Western blotting at the indicated time points. The panels show representative data from four biological replicates. B , expression of PIM1, PIM2, and PIM3 in activated human peripheral CXCR3+ and CXCR3− CD4+ T cells. CD4+ cells were isolated from buffy coats, and the CXCR3-positive and -negative populations were isolated by flow cytometric cell sorting. Sorted cells were activated and harvested at the indicated time points. The panels show representative data of five biological replicates. C , CD4+ T cells were nucleofected with STAT4 (pooled siRNAs 1 + 2) or NT siRNA and polarized in the Th1 direction 20–24 h after nucleofection. Samples were harvested for Western blotting at the indicated time points. The panels show representative data from three biological replicates. D , CD4+ T cells were nucleofected with STAT6 or NT siRNA and polarized in the Th2 direction 24 h after nucleofection. Samples were harvested for Western blotting at the indicated time points. The panels show representative data from two biological replicates. Vertical lines represent repositioned gel lanes that are from the same blot and the same exposure. d , days.

Article Snippet: Proteins were detected using the following antibodies: mouse α-PIM1 (12H8, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit α-PIM2 (ATLAS Antibodies, Stockholm, Sweden or D1D2, Cell Signaling Technology, Beverly, MA), rabbit α-PIM3 (D17C9, Cell Signaling Technology), mouse α-T-BET (4B10, Santa Cruz Biotechnology), rabbit α-STAT1 p84/p91 (E-23, Santa Cruz Biotechnology), rabbit α-STAT4 (C-20, Santa Cruz Biotechnology), mouse α-STAT6 (BD Biosciences), mouse α-GAPDH (number 5G4, 6C5, HyTest, Turku, Finland), and mouse α-β-actin (Sigma-Aldrich).

Techniques: Expressing, Knockdown, Cell Differentiation, Isolation, Western Blot, FACS

Journal: The Journal of Biological Chemistry

Article Title: Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation *

doi: 10.1074/jbc.M112.361709

Figure Lengend Snippet: PIM kinases regulate the IL-12R β 2 / STAT4 pathway in Th1-polarized cells. CD4+ cells were nucleofected and cultured as in . A , samples were harvested at different time points during the culture for TaqMan RT-PCR analysis. These graphs represent the average -fold differences ±S.E. ( error bars ) of IL-12R β 2 mRNA levels in PIM siRNA-treated Th1 cells compared with NT siRNA-treated Th1 cells. Data were calculated from five of six independent experiments. B , expression of the cell surface IL-12Rβ2 protein was analyzed by immunofluorescent staining and flow cytometry at the indicated time points. The bars represent the average -fold changes ±S.E. ( error bars ) of the geometric mean intensities of IL-12Rβ2 in PIM siRNA samples compared with control samples. The data were calculated from six independent experiments. The histograms show representative data from these experiments ( red line , NT siRNA Th1; blue line , PIM123 siRNA Th1; gray lines , isotype controls). Samples were harvested for Western blotting ( C ) and TaqMan RT-PCR ( D ) analyses at the indicated time points. The bars represent the mean values ±S.E. ( error bars ) of the relative levels of STAT4 between control and PIM siRNA samples obtained by quantifying and normalizing against the levels of β-actin. The value of control samples was set as 1. The data were calculated from four independent experiments. The Western blot of STAT1 shows representative data from four independent experiments. Vertical lines represent repositioned gel lanes that are from the same blot and the same exposure. A–D , statistical significances were calculated using the two-tailed paired t test: *, p < 0.05; **, p < 0.02; ***, p > 0.01 ( A , C , and D ); *, p < 0.005; **, p < 0.0005 ( B ). NT indicates control siRNA. Rel. prot. , relative protein; PerCP , peridinin-chlorophyll protein complex.

Article Snippet: Proteins were detected using the following antibodies: mouse α-PIM1 (12H8, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit α-PIM2 (ATLAS Antibodies, Stockholm, Sweden or D1D2, Cell Signaling Technology, Beverly, MA), rabbit α-PIM3 (D17C9, Cell Signaling Technology), mouse α-T-BET (4B10, Santa Cruz Biotechnology), rabbit α-STAT1 p84/p91 (E-23, Santa Cruz Biotechnology), rabbit α-STAT4 (C-20, Santa Cruz Biotechnology), mouse α-STAT6 (BD Biosciences), mouse α-GAPDH (number 5G4, 6C5, HyTest, Turku, Finland), and mouse α-β-actin (Sigma-Aldrich).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Flow Cytometry, Control, Western Blot, Two Tailed Test

Journal:

Article Title: IFN-? Is Not Sufficient to Drive Th1 Development Due to Lack of Stable T-bet Expression 1

doi:

Figure Lengend Snippet: A and B, Human PBMCs were activated and expanded for two consecutive weeks with PHA in the presence of IL-2 and IL-12. On day 14, cells were rested in fresh medium for 30 minutes and stimulated with either IL-12 or IFN-αA for the indicated periods of time. Immunoprecipitation was performed using polyclonal antisera for human STAT4 or STAT1, and Western blotting was performed for phosphorylated STAT4 or STAT1, as indicated. Membranes were then stripped and re-probed for total STAT4 or STAT1. A, Time course of STAT4 and STAT1 phosphorylation in human Th1 PBMCs stimulated with IL-12 (lanes 2–7) or IFN-αA (lanes 8–13) for 0–24 hours. B, Time course of STAT4 phosphorylation in human Th1 PBMCs stimulated with IL-12 (lanes 2–7) or IFN-αA (lanes 8–13) for 0–6 hours. C through G, Freshly isolated PBMCs were activated with medium alone, IL-12, or IFN-α at various time points up to 24 hours. Cells were stained for CD4, CD45RA, and intracellular STAT4 and phosphotyrosine STAT4 (P-Y STAT4) as described in the Materials and Methods. C and D, Total STAT4 (C) and P-Y STAT4 (D) were measured from cells activated for 30 min by flow cytometry. Data are gated on live, CD4+, and CD45RA+ cells. Black line, unstimulated; green line, IL-12 stimulated; red line, IFN-α stimulated; gray shaded, non-immune rabbit Ig control. E, Gating scheme is shown for the analysis of CD4+, CD45RA- (R2) and CD45RA+ (R3) cells. F and G, CD45RA- (F) and CD45RA+ (G) gated cells are represented as a percentage of cells that display increased P-Y STAT4 staining over a 24 hour period. Open square, unstimulated; open triangle, IL-12 stimulated; open circle, IFN-α stimulated. H and I, Human PBMCs were activated and stained as above and analyzed on the ImageStream® imaging flow cytometer. Single cell images were gated on the live, CD4+, and CD45RA+ populations. H, Cells displaying positive staining for P-Y STAT4 were categorized for either low similarity (left panel) or high similarity (right panel) of co-localization of P-Y STAT4 within the nucleus based on nuclear dye staining (Draq-5). I, Cells displaying elevated P-Y STAT4 staining were quantified for co-localization of P-Y STAT4 within the nucleus of cells treated with IL-12 (closed square) or IFN-α (closed triangle).

Article Snippet: Polyclonal rabbit α-human STAT4, α-human STAT1, and α-human T-bet antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Isolation, Staining, Flow Cytometry, Control, Imaging

Journal:

Article Title: IFN-? Is Not Sufficient to Drive Th1 Development Due to Lack of Stable T-bet Expression 1

doi:

Figure Lengend Snippet: (A) Spleen and lymph node cells from DO11.10 mice were activated with OVA peptide under Th1-inducing conditions and transduced with retrovirus vectors expressing GFP alone (GFPRV) or the full-length mIFNAR2 subunit. Cells were sorted on day 7 based on GFP expression and restimulated with irradiated BALB/c splenocytes and OVA peptide. Following expansion for an additional 7 days, resting cells were activated with either IL-12 or IFN-α for the times indicated in the figure. Cells were then stained and analyzed for intracellular tyrosine-phosphorylated STAT4 as described in Fig. 5. (B and C) Day 14 transduced Th1 cells were activated with either IL-12 + IL-18, IFN-α + IL-18, or with the individual cytokines indicated in the figure for 24 hours. Brefelden A was added during the last 4 hours of stimulation. Cells were then stained for mCD4 and IFN-γ and analyzed by flow cytometry. Data were gated on live cells and GFP expression.

Article Snippet: Polyclonal rabbit α-human STAT4, α-human STAT1, and α-human T-bet antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transduction, Expressing, Irradiation, Staining, Flow Cytometry