total phosphorylated stat4 site 693  (R&D Systems)

Journal: Experimental Hematology & Oncology

Article Title: Improving CAR-T cell function through a targeted cytokine delivery system utilizing car target-modified extracellular vesicles

doi: 10.1186/s40164-025-00701-z

Figure Lengend Snippet: IL-12 EVs enhance the function of CAR-T cells. ( A ) CAR-T cells were treated with control EVs or IL-12 EVs for 30 min, and the phosphorylation of STAT4 was analyzed by western blotting. Experiments are representative of 3 independent repeats with similar results. (B-G) CAR-T cells (cell density: 1*10 6 /mL) were mixed with 5 × 10 4 Raji cells at an effector-to-target ratio of 1:1 and treated with PBS, control EVs, rhIL-12, and IL-12 EVs repectively. Protein concentration of EVs is 167 µg/mL, and rhIL-12 concentration is 667 pg/mL. ( B , C ) Cytokine secretion by CAR-T cells was detected by ELISA after 24 h of coculture ( n = 3 donors). ( D ) The proportion of CAR-T cells was analyzed by flow cytometry after 72 h of coculture ( n = 3 donors). ( E ) CD107a expression in CD8 + CAR-T cells was detected by flow cytometry ( n = 3 donors). ( F ) Raji cell death was determined using PI (BD Pharmingen) and analyzed by using flow cytometry after 24 h ( n = 5 donors). ( G ) Subsets were detected via flow cytometry in CAR-T cells after 7 days of treatment ( n = 3 donors). Data are presented as mean ± SEM. Statistical analysis was performed using one-way or two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Total STAT4 and phosphorylated STAT4 in T cells were detected using an anti-Stat4 (C46B10) antibody (CST, 2653 S, 1:1000) and an anti-phospho-Stat4 (Tyr693) antibody (CST, 5267 S, 1:1000).

Techniques: Control, Phospho-proteomics, Western Blot, Protein Concentration, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing

Journal: Journal of Veterinary Medicine

Article Title: Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells

doi: 10.1155/2017/5701016

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Phosphorylated Human Stat4 (pY693) Peptide , Stat4 , Mouse IgG2b, κ , Alexa Fluor 647 , BD Biosciences , 38/p-Stat4 , 1 : 50.

Techniques:

Journal: Journal of Veterinary Medicine

Article Title: Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells

doi: 10.1155/2017/5701016

Figure Lengend Snippet: Comparison of protein phosphorylation of mADSCs and cADSCs. The red peaks represent the isotype controls, and the blue peaks represent antigens. β -catenin, β -catenin (pS45), Akt (pS473), Stat3, Stat4, Stat5, and ERK1/2 were detected, whereas Akt (pT308), CD140b, Stat1, Stat6, p38 MAPK, and Smad2/3 were not detected in mADSCs. In addition, β -catenin, β -catenin (pS45), Akt (pS473), and Stat4 were detected, whereas Akt (pT308), CD140b, Stat1, Stat3, Stat5, Stat6, p38 MAPK, Smad2/3, and ERK1/2 were negative in cADSCs. The percentages shown in the figure represent the positive rates of the antigen.

Article Snippet: Phosphorylated Human Stat4 (pY693) Peptide , Stat4 , Mouse IgG2b, κ , Alexa Fluor 647 , BD Biosciences , 38/p-Stat4 , 1 : 50.

Techniques:

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: bioRxiv

Article Title: Dendritic cell Piezo1 integrating mechanical stiffness and inflammatory signals directs the differentiation of T H 1 and T reg cells in cancer

doi: 10.1101/2022.05.17.492270

Figure Lengend Snippet: ( A ) Expression of TGFβR2 and IL-12Rβ2 in T cells cocultured with WT or Piezo1 ΔDC splenic DCs for 5 days. A representative figure is shown on the left, and the data are summarized on the right. ( B ) Intracellular staining of p-Smad3 and p-STAT4 in T cells cocultured with WT or Piezo1 ΔDC splenic DCs for 5 days. A representative figure is shown on the left, and the data are summarized on the right. ( C-D ) Sorted naïve T cells were transfected with control, TGFβR2 shRNA vector or IL-12Rβ2 shRNA vector and cocultured with WT or Piezo1 ΔDC DCs for 5 days. Intracellular staining of IFNγ (C; upper panel) and Foxp3 (C; lower panel) in T cells. A representative figure is shown on the left, and the data are summarized on the right. (D) Intracellular staining of p-Smad3 and p-STAT4 in T cells. Data are summarized. The data are representative of three to four independent experiments (mean ± s.d.; n=4). *** P <0.001, compared with the indicated groups.

Article Snippet: For the detection of phosphorylated signaling proteins, purified cells were activated with LPS (Sigma), immediately fixed with Phosflow Perm buffer (BD Biosciences) and stained with phycoerythrin or allophycocyanin directly conjugated to antibodies against Erk phosphorylated at Thr202 and Tyr204 (20A; Cat#612566, RRID:AB_399857; BD Biosciences), p38MAPK phosphorylated at Thr180 and Thr182 (D3F9; Cell Signaling Technology), JNK phosphorylated at Thr183 and Tyr185 (G9; Cell Signaling Technology), STAT4 phosphorylated at Tyr701 and Ser727 (58D6; Cell Signaling Technology), and SMAD3 phosphorylated at Tyr705 and Ser727 (D3A7; Cell Signaling Technology), as described previously( ).

Techniques: Expressing, Staining, Transfection, shRNA, Plasmid Preparation