rabbit anti-phosphorylated stat4 antibody  (Cell Signaling Technology Inc)

Journal: Arthritis Research & Therapy

Article Title: Natural killer cells control a T-helper 1 response in patients with Behçet's disease

doi: 10.1186/ar3005

Figure Lengend Snippet: IL-12 signaling of natural killer cells in inactive Behçet's disease patients . IL-12-induced signal transducer and activator of transduction 4 (Stat4) phosphorylation in natural killer (NK) cells from inactive Behçet's disease (iBD) patients and healthy controls. (a) NK cells from six iBD patients and five healthy controls were stimulated with IL-12, and were subjected to immunoblotting for the detection of phosphorylated Stat4 (pStat4) and total Stat4. (b) Phosphorylation status of Stat4, which is expressed as the ratio of the intensity of pStat4 to that of total Stat4, in NK cells from six iBD patients and five healthy controls. Horizontal bars, mean values.

Article Snippet: The antibodies used were rabbit anti-phosphorylated-Stat4 polyclonal antibodies (Zymed Laboratories, South San Francisco, CA, USA) and rabbit anti-Stat4 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transduction, Western Blot

Journal: International journal of molecular sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223.

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Figure 1. Endogenous expression of IL-23p19 and clinical assessments in the LVshIL-23p19-treated mice with collagen-induced arthritis (CIA). (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis of IL-23p19 expression in the synovial tissue of mice with CIA (Day 10, 21, 42). Each lane represents pooled samples from 3 animals. Results are representative of two independent experiments. (B) Amelioration in mice with CIA by evaluating with arthritis score after the LVshIL- 23p19 treatment. Mice that had been immunized with collagen on days 0 and 21 were injected intraperitoneally with LVshIL-23p19, LVshLuc and medium on day 21, respectively. The arrow indicates the time at which the lentiviral vectors were injected. Each value shown represents the mean ± SEM (n = 6). (C) Representative images of the ankle sections by hematoxylin and eosin staining on day 45. (D) Representative images and quantification of the levels of phosphorylated signal transducers and activators of transcription 4 (p-STAT4) by immunohistochemical staining on day 45. Each value shown represents the mean ± SEM (n = 3). (E) Representative images of the levels of miR-223 by in situ hybridization on day 45. Scale bars represent 500, 200 and 50 µm in ×40, ×100 and ×400 magnifications, respectively. Each value shown represents the mean ± SEM (n = 3). Black boxed areas were shown at higher magnification in the panels (×400) beneath them. Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Immunohistochemical staining, In Situ Hybridization

Journal: International journal of molecular sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223.

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Figure 2. Association of IL-23 signaling with miR-223 in osteoclastogenesis. (A) Tartrate-resistant acid phosphatase (TRAP) staining and quantification in IL-23 (5 and 20 ng/mL) treated bone marrow- derived macrophages (BMMs). Each value shown represents the mean ± SEM (n = 4). (B) TRAP stain- ing and quantification in LVmiR-223T and LVshLuc-infected BMMs (MOI = 0.05 and 0.5) in response to IL-23 stimulation (20 ng/mL). Each value shown represents the mean ± SEM (n = 3). (C) TRAP staining and quantification in LVshSTAT4#1 (TRCN0000081638), LVshSTAT4#2 (TRCN0000081639) and LVshLuc-infected BMMs (MOI = 1 and 5) in response to IL-23 stimulation (20 ng/mL). Scale bars represent 200 µm in ×100 magnifications. Each value shown represents the mean ± SEM (n = 3). (D) Binding of phosphorylated STAT4 to the miR-223 promoter in IL-23-treated RAW264.7 cells by determining with quantitative chromatin immunoprecipitation (qChIP) assay. Each value shown represents the mean ± SEM (n = 3). (E) Expression of miR-223 in BMMs treated with vari- ous concentrations of IL-23 for 12 h, as determined by quantitative RT-PCR (qRT-PCR). Each value shown represents the mean ± SEM (n = 3). (F) Expression of miR-223 in LVshLuc, LVshSTAT4#1 (TRCN0000081638) and LVshSTAT4#2 (TRCN0000081639)-transduced BMMs upon stimulating with IL-23 (20 ng/mL) for 12 h, as determined by qRT-PCR. Each value shown represents the mean ± SEM (n = 3). Red boxed areas are shown at higher magnification in the panels beneath them. Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Staining, Derivative Assay, Infection, Binding Assay, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223.

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Figure 4. A schematic overview of silencing IL-23-STAT4-miR-223 signaling axis in preventing osteoclastogenesis. IL-23p19 can bind to the IL-23 receptor (IL-23R), which causes phosphorylation and translocation of STAT4 into the nucleus. P-STAT4 further conjugates with the promoter region of primary miR-223 (pri-miR-223) and the transactivated pri-miR-223 translocates into the cytosol to become mature miR-223 (miR-223) and mediates osteoclastogenesis. The signaling axis, IL-23- STAT4-miR-223 is proved to mediate osteoclastogenesis by introducing IL-23-stimulated BMMs and mouse CIA model with LVshIL-23p19, LVshSTAT4, LSF, and LVmiR-223T that target IL-23p19, STAT4, and miR-223.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Phospho-proteomics, Translocation Assay

Journal:

Article Title: A NOVEL VIP SIGNALING PATHWAY IN T CELLS cAMP→ Protein Tyrosine Phosphatase (SHP-2?)→JAK2/STAT4→Th1 differentiation

doi: 10.1016/j.peptides.2007.03.015

Figure Lengend Snippet: (A) CD4+ T cells were stimulated with immobilized anti-CD3 and anti-CD28 plus IL-2 for 48 hr. After extensive washing, the cells were replated in serum-free medium for 24hr, treated with VIP for 30 min, followed by IL-12 (100ng/ml) for 10 min. The cells were permeabilized and stained for P-STAT4 and total STAT4. (B). Cells treated as in (A) were preincubated with different concentration of VIP (10−6 -10−10 M) for 30 min, followed by IL-12 (100ng/ml) for 10 min. (C). CD4+ T cells were treated as in (A). VIP (10-7M) was added at different times: 10 min before IL-12, same time as IL-12, 5 min after IL-12. One experiment out of three is shown.

Article Snippet: The following antibodies were used for FACS analysis: PE-conjugated rat anti-mouse IFNγ and PE-conjugated rat IgG1(PharMingen), rabbit IgG (Sigma), rabbit anti-STAT4 and rabbit anti-phosphorylated (Y693)-STAT4 (Zymed), phospho-Jak2 (Tyr 1007/1008) antibody, rabbit polyclonal anti-mouse SHP-2, rabbit polyclonal anti-mouse phosphorylated SHP-2 (Cell Signaling) and FITC-conjugated goat anti-rabbit IgG (Sigma).

Techniques: Staining, Concentration Assay

Journal:

Article Title: A NOVEL VIP SIGNALING PATHWAY IN T CELLS cAMP→ Protein Tyrosine Phosphatase (SHP-2?)→JAK2/STAT4→Th1 differentiation

doi: 10.1016/j.peptides.2007.03.015

Figure Lengend Snippet: (A). Cells were cultured as in Fig. 2A. Following replating in serum-free conditions for 24h, the cultures were treated with VIP (10-7M), dbcAMP (10−4 M) or forskolin (10−5 M) for 30 min, followed by IL-12 for 10 min and intracellular staining for P-STAT4. (B). Cultures were pretreated with H89 (10−6 M) for 15 min, followed by VIP (10-7M) for 30 min and IL-12 for 10 min. The cells were stained for intracellular P-STAT4. One representative experiment out of three is shown.

Article Snippet: The following antibodies were used for FACS analysis: PE-conjugated rat anti-mouse IFNγ and PE-conjugated rat IgG1(PharMingen), rabbit IgG (Sigma), rabbit anti-STAT4 and rabbit anti-phosphorylated (Y693)-STAT4 (Zymed), phospho-Jak2 (Tyr 1007/1008) antibody, rabbit polyclonal anti-mouse SHP-2, rabbit polyclonal anti-mouse phosphorylated SHP-2 (Cell Signaling) and FITC-conjugated goat anti-rabbit IgG (Sigma).

Techniques: Cell Culture, Staining

Journal:

Article Title: A NOVEL VIP SIGNALING PATHWAY IN T CELLS cAMP→ Protein Tyrosine Phosphatase (SHP-2?)→JAK2/STAT4→Th1 differentiation

doi: 10.1016/j.peptides.2007.03.015

Figure Lengend Snippet: (A). Cells were cultured as in Fig. 2A. Following replating in serum-free conditions for 24h, the cultures were preincubated with bpV (phen) (100 μM) (A), Na3VO4 (1mM) (B), or okadaic acid (OA, 50 nM) (C) for 15 min, followed by VIP (10−7 M) for 30 min and IL-12 for 10 min. The permeabilized cells were stained for intracellular p-STAT4. Pne experiment out of four is shown.

Article Snippet: The following antibodies were used for FACS analysis: PE-conjugated rat anti-mouse IFNγ and PE-conjugated rat IgG1(PharMingen), rabbit IgG (Sigma), rabbit anti-STAT4 and rabbit anti-phosphorylated (Y693)-STAT4 (Zymed), phospho-Jak2 (Tyr 1007/1008) antibody, rabbit polyclonal anti-mouse SHP-2, rabbit polyclonal anti-mouse phosphorylated SHP-2 (Cell Signaling) and FITC-conjugated goat anti-rabbit IgG (Sigma).

Techniques: Cell Culture, Staining

Journal:

Article Title: A NOVEL VIP SIGNALING PATHWAY IN T CELLS cAMP→ Protein Tyrosine Phosphatase (SHP-2?)→JAK2/STAT4→Th1 differentiation

doi: 10.1016/j.peptides.2007.03.015

Figure Lengend Snippet: VIP acting through the VPAC1/2/ receptors activates adenylate cyclase resulting in the activation of PKA. PKA phosphorylates SHP-2, a protein tyrosine phosphatase which in turn acts on Jak2 and possibly directly on STAT4. Dephosphorylation of Jak2 and STAT4 interrupts the IL-12 signaling pathway and prevents the expression of genes involved in Th1 differentiation and function.

Article Snippet: The following antibodies were used for FACS analysis: PE-conjugated rat anti-mouse IFNγ and PE-conjugated rat IgG1(PharMingen), rabbit IgG (Sigma), rabbit anti-STAT4 and rabbit anti-phosphorylated (Y693)-STAT4 (Zymed), phospho-Jak2 (Tyr 1007/1008) antibody, rabbit polyclonal anti-mouse SHP-2, rabbit polyclonal anti-mouse phosphorylated SHP-2 (Cell Signaling) and FITC-conjugated goat anti-rabbit IgG (Sigma).

Techniques: Activation Assay, De-Phosphorylation Assay, Expressing