mouse p-stat3 (y705) antibody  (Cell Signaling Technology Inc)

Journal: Experimental & Molecular Medicine

Article Title: mtSTAT3 suppresses rheumatoid arthritis by regulating Th17 and synovial fibroblast inflammatory cell death with IL-17-mediated autophagy dysfunction

doi: 10.1038/s12276-024-01376-y

Figure Lengend Snippet: a Autophagosomes and autophagolysosomes were analyzed in OA and RA FLSs via transmission electron microscopy (scale bars = 0.5 μm, AP autophagosome, AL autophagolysosome). b RA FLSs were cultured with 10 μM rapamycin and 20 μM chloroquine for 24 h. Autophagosomes and autophagolysosomes were analyzed with anti-LC3-APC antibodies, anti-p62-FITC antibodies, anti-LAMP1-PE antibodies, and DAPI via confocal microscopy. c Mitochondria were isolated from IL-17-stimulated RA FLSs. The mitochondrial lysates were analyzed via western blotting with anti- p- STAT3 Y705 , anti- p- STAT3 S727 , anti-STAT3, anti-tubulin, and anti-COX4 antibodies. d RA FLSs were cultured with IL-17 for 12 h and then lysed to obtain proteins. The expression levels of MFN2, DRP1, and p- DRP1, which are dynamic mitochondrial molecules, were measured. e RA FLSs were transfected with mock or MLS-STAT3 vectors. P- STAT3 S727 expression in cytoplasmic lysates and mitochondrial lysates was analyzed via western blotting. f RA FLSs were transfected with mock or MLS-STAT3 DNA vectors. The cells were cultured with IL-17 for 24 h posttransfection. Autophagosomes and autophagolysosomes were analyzed via confocal microscopy using anti-LC3-APC, anti-p62-FITC, and anti-LAMP1-PE antibodies and DAPI. g RA FLSs were cultured with IL-17 (10 ng/mL) for 24 h posttransfection. Inflammatory cell death molecules in the cell lysates were analyzed by western blotting. All the experiments were performed in triplicate. Bars represent the mean ± standard deviation (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

Article Snippet: Primary antibodies against RIP1, RIP3, p -MLKL, p -STAT3 S727 , p -STAT3 Y705 , STAT3, Mfn2, DRP1, p -DRP1, LC3, p62, GAPDH, tubulin, Cox4, or β-actin (Cell Signaling Technology) were diluted at a concentration of 1:500–1:1000.

Techniques: Transmission Assay, Electron Microscopy, Cell Culture, Confocal Microscopy, Isolation, Western Blot, Expressing, Transfection, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: mtSTAT3 suppresses rheumatoid arthritis by regulating Th17 and synovial fibroblast inflammatory cell death with IL-17-mediated autophagy dysfunction

doi: 10.1038/s12276-024-01376-y

Figure Lengend Snippet: a Schematic map of the mouse MLS-STAT3-FLAG vector. b Western blot of mitochondria from mock- and MLS-STAT3-transfected NIH3T3 cells. Lanes were derived from the same gel and rearranged. c Schematic map of the schedule for arthritis induction. A total of 100 μg of mock or MLS-STAT3 DNA vector was injected every 10 days for 9 weeks after the first immunization. The severity scores and incidence of arthritis were analyzed every week. d , e Ankle joints were obtained 9 weeks after the first immunization, and the tissues were stained with H&E or antibodies against IL-17, IL-6, TNF-α, IL-1β, RIP1, RIP3, or p -MLKL. Representative images (scale bar = 100 μm) are displayed as the number of positive cells (dark brown) in five mice from each group. All the experiments were performed in triplicate. Arthritis scores are displayed as the mean ± standard error of the mean, and the bars represent the mean ± standard deviation (* p < 0.05, ** p < 0.01).

Article Snippet: Primary antibodies against RIP1, RIP3, p -MLKL, p -STAT3 S727 , p -STAT3 Y705 , STAT3, Mfn2, DRP1, p -DRP1, LC3, p62, GAPDH, tubulin, Cox4, or β-actin (Cell Signaling Technology) were diluted at a concentration of 1:500–1:1000.

Techniques: Plasmid Preparation, Western Blot, Transfection, Derivative Assay, Injection, Staining, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: mtSTAT3 suppresses rheumatoid arthritis by regulating Th17 and synovial fibroblast inflammatory cell death with IL-17-mediated autophagy dysfunction

doi: 10.1038/s12276-024-01376-y

Figure Lengend Snippet: a – c Mock- or MLS-STAT3-injected mice were sacrificed at 9 weeks after the first immunization. a Splenic tissues were stained with specific antibodies for the analysis of Th17 cells (anti-IL-17-PE and anti-CD4-FITC), Tregs (anti-Foxp3-PE, anti-CD25-APC, and anti-CD4-FITC), and mitochondrial STAT3 (anti- p -STAT3 S727 -PE, anti-CD4-FITC, and anti-Cox4-APC). Representative numbers of positive cells from five different tissues are shown (left). b The expression levels of RIP3, p- MLKL, and GAPDH were measured in splenocyte lysates from the mice. c Splenic tissues were stained with anti-LC3-FITC, anti-p62-PE, and anti-LAMP1-PE antibodies and DAPI for autophagosome and autophagolysosome analysis. Bars represent the mean ± standard deviation (* p < 0.05).

Article Snippet: Primary antibodies against RIP1, RIP3, p -MLKL, p -STAT3 S727 , p -STAT3 Y705 , STAT3, Mfn2, DRP1, p -DRP1, LC3, p62, GAPDH, tubulin, Cox4, or β-actin (Cell Signaling Technology) were diluted at a concentration of 1:500–1:1000.

Techniques: Injection, Staining, Expressing, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: mtSTAT3 suppresses rheumatoid arthritis by regulating Th17 and synovial fibroblast inflammatory cell death with IL-17-mediated autophagy dysfunction

doi: 10.1038/s12276-024-01376-y

Figure Lengend Snippet: a – e 100 μg of the 705 mutant (STAT3 Y705F ) or the 705 mutant or the MLS-STAT3 DNA vector was injected every 10 days for 9 weeks after the first immunization. a Severity scores and incidence of arthritis were analyzed every week. b Ankle joints were obtained at 9 weeks after the first immunization, and the tissues were stained with H&E and safranin O for analysis of inflammation and bone damage. c Populations of IFN-γ + , IL-4 + , IL-17 + , and CD25 + Foxp3 + cells among splenic CD4 + T cells were analyzed by flow cytometry. d The expression levels of RIP1, RIP3, p- MLKL, and GAPDH were measured in splenocyte lysates from each group. Lanes were derived from the same gel and rearranged. e Ankle joint tissues were stained with antibodies against IL-17, IL-6, TNF-α, and IL-1β. Representative images (scale bar = 100 μm) are displayed as the number of positive cells (dark brown) in three mice from each group. All the experiments were performed in triplicate. Arthritis scores are displayed as the mean ± standard error of the mean, and the bars represent the mean ± standard deviation (* p < 0.05, ** p < 0.01).

Article Snippet: Primary antibodies against RIP1, RIP3, p -MLKL, p -STAT3 S727 , p -STAT3 Y705 , STAT3, Mfn2, DRP1, p -DRP1, LC3, p62, GAPDH, tubulin, Cox4, or β-actin (Cell Signaling Technology) were diluted at a concentration of 1:500–1:1000.

Techniques: Mutagenesis, Plasmid Preparation, Injection, Staining, Flow Cytometry, Expressing, Derivative Assay, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: mtSTAT3 suppresses rheumatoid arthritis by regulating Th17 and synovial fibroblast inflammatory cell death with IL-17-mediated autophagy dysfunction

doi: 10.1038/s12276-024-01376-y

Figure Lengend Snippet: a – f Seven days after CIA induction, ZnSO 4 (5 mg/kg) was orally administered for 9 weeks. a Severity scores and incidence of arthritis were analyzed every week. b Ankle joints were obtained at 9 weeks, and the tissues were stained with H&E and safranin O for analysis of inflammation and bone damage. c Splenic tissues were stained with specific antibodies for the analysis of Th17 cells (anti-IL-17-PE and anti-CD4-FITC) and mitochondrial STAT3 (anti- p -STAT3 S727 -PE, anti-CD4-FITC, and anti-Cox4-APC). d Splenocytes were stained with anti-CD4 and anti-IL-17 antibodies at 9 weeks after the first immunization and analyzed by flow cytometry. e The sectioned ankle joint tissues were immunohistochemically stained to identify cells positive for IL-6, IL-1β, IL-17, TNF-α, RIP1, and RIP3. Representative images (scale bar = 100 μm) are displayed as the number of positive cells (dark brown) in five mice per group. f Splenic tissues were stained with anti-LC3-FITC, anti-p62-PE, and anti-LAMP1-PE antibodies and DAPI for autophagosome and autophagolysosome analysis. Arthritis scores are displayed as the mean ± standard error of the mean, and the bars represent the mean ± standard deviation (* p < 0.05, ** p < 0.01, **** p < 0.001).

Article Snippet: Primary antibodies against RIP1, RIP3, p -MLKL, p -STAT3 S727 , p -STAT3 Y705 , STAT3, Mfn2, DRP1, p -DRP1, LC3, p62, GAPDH, tubulin, Cox4, or β-actin (Cell Signaling Technology) were diluted at a concentration of 1:500–1:1000.

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: International journal of molecular sciences

Article Title: IL-6 Does Not Influence the Expression of SLC41A1 and Other Mg-Homeostatic Factors.

doi: 10.3390/ijms252413274

Figure Lengend Snippet: Figure 3. Immunodetection of post-translational modifications of the STAT3 protein triggered by the application of hIL-6 to the HEK-Blue™IL-6 cell line. (a) Cells were treated with 1 or 100 ng/mL of hIL-6 for 3 or 6 h. (b) After 1 h of pretreatment with 10 µmol/L Stattic, 100 ng/mL hIL-6 was added to the culture medium, and the cells were incubated for another hour. The application of a Stattic inhibitor suppressed the IL-6-induced phosphorylation of Y705 and the subsequent acetylation of K685 of the STAT3 molecule.

Article Snippet: Antibodies against total STAT3 sc-8019 (1:500), phosphorylated Y705 sc-81523 (1:500), phosphorylated S727 sc-71792 (1:500) (all Santa Cruz Biotechnology, USA), or acetylated K685 PA5-17429 (1:1000) (Invitrogen, Carlsbad, CA, USA) were used, followed by a 1-h incubation with either anti-mouse (1:10,000) or antirabbit (1:20,000) horseradish peroxidase-conjugated secondary antibody (both Sigma-Aldrich, Louisville, KY, USA).

Techniques: Immunodetection, Incubation, Phospho-proteomics

Journal: International journal of molecular sciences

Article Title: IL-6 Does Not Influence the Expression of SLC41A1 and Other Mg-Homeostatic Factors.

doi: 10.3390/ijms252413274

Figure Lengend Snippet: Figure 4. Treatment of HEK-Blue™IL-6, HepG2, U-266, and PANC-1 cell lines with 50 ng/mL hIL-6 for 30 min or 3 h triggered STAT3 phosphorylation at Y705. In the case of HepG2 and U-266, S727

Article Snippet: Antibodies against total STAT3 sc-8019 (1:500), phosphorylated Y705 sc-81523 (1:500), phosphorylated S727 sc-71792 (1:500) (all Santa Cruz Biotechnology, USA), or acetylated K685 PA5-17429 (1:1000) (Invitrogen, Carlsbad, CA, USA) were used, followed by a 1-h incubation with either anti-mouse (1:10,000) or antirabbit (1:20,000) horseradish peroxidase-conjugated secondary antibody (both Sigma-Aldrich, Louisville, KY, USA).

Techniques: Phospho-proteomics