rabbit anti phospho stat3 py705  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Structures of human protein tyrosine phosphatase variants reveal targetable allosteric sites

doi: 10.1016/j.jbc.2025.110852

Figure Lengend Snippet: Functional characterization of PTP1B mutants in cells. A , mapping of variants identified in the STILTS cohort and UK Biobank on the primary structure of PTP1B (proline [Pro]-rich region; endoplasmic reticulum [ER] anchor). B , representative confocal fluorescence microscopy images showing protein localization of WT/mutant PTP1B in HEK293 cells. Blue : DAPI (nuclei), red : Alexa 647 for HA-tagged PTP1B, green : Alexa 488 for PDI, yellow : DyLight Phalloidin 554. The scale bar represents 50 μm. C , effect of WT/mutant PTP1B on leptin-stimulated STAT3 phosphorylation (Tyr705). n = 4 to 7; data expressed as mean ± SEM normalized to WT (0%) and WT leptin-stimulated (100%) (AU, arbitrary units). Two-tailed unpaired one-sample t test on log-transformed data for mutant versus WT. D , effect of WT/mutant PTP1B on leptin-induced POMC transcription in a luciferase reporter assay. n = 5 to 9; data expressed as mean ± SEM relative to WT. Two-tailed unpaired one-sample t test for mutant versus WT. E , PTP1B mutations categorized as loss of function (LOF; blue ), WT-like (WT; gray ) or gain of function (GOF; red ) based on phosphorylation and localization assays presented in and . Statistically significant difference between mutant and WT (expressed as % WT) in leptin-stimulated STAT3 phosphorylation shown. Data are log transformed; values <10% are set to 10% for visualization. Median shown ( box shows interquartile range [IQR]; whiskers extend to 1.5∗IQR). Results were analyzed with an unpaired single-sample t test . PTP1B, protein tyrosine phosphatase 1B; STILTS, Study Into Lean and Thin Subjects.

Article Snippet: Membranes were blocked with 3% bovine serum albumin (BSA) solution in Tris-buffered saline with Tween-20 (TBS-T) for 1 h at room temperature and probed overnight at 4 °C using Rabbit anti-STAT3 at 1:1000 dilution (Cell Signaling Technology; 12640), Rabbit anti-Phospho STAT3 (pY705) (Cell Signaling Technology; 9145) at 1:1000 dilution, Rabbit anti-HA (Cell Signaling Technology; C29F4) at 1:1000 dilution, Mouse anti-DYK (FLAG) tag M2 antibody (Sigma; F1804), Rabbit anti-phospho AKT (pS473) (Cell Signaling Technology; 4060) at 1:1000 dilution, Rabbit anti-AKT pan (Cell Signaling Technology; 4691) at 1:1000 dilution, Rabbit anti-β-actin (Cell Signaling Technology; 4967) at 1:1000 dilution, and Rabbit anti-vinculin (Abcam; ab129002), all prepared in the blocking buffer.

Techniques: Functional Assay, Fluorescence, Microscopy, Mutagenesis, Phospho-proteomics, Two Tailed Test, Transformation Assay, Luciferase, Reporter Assay

Journal: bioRxiv

Article Title: G1899, an American ginseng extract, alleviates neuroinflammation and cognitive impairment in models of Alzheimer’s disease

doi: 10.1101/2025.09.04.674314

Figure Lengend Snippet: G1899 promotes microglial homeostasis and attenuates detrimental microglial activation induced by amyloid β oligomer (Aβo). a Schematic illustration (created with BioRender.com) of the treatment time window for G1899 and Aβo in mouse primary microglia. b Representative immunoblot images showing dose-dependent upregulation of anti-TMEM119 (homeostatic marker) and anti-CD68 (phagocytic marker) in G1899-treated microglia. Quantification of c TMEM119 and d CD68 normalized to β-actin. e Effects of G1899 on NLRP3 inflammasome formation and STAT3 signaling, with quantification of f NLRP3, g Caspase-1, h ASC, i pY705 STAT3, j pS727 STAT3, and k total STAT3 normalized to β-actin. l Immunofluorescence analysis of morphological changes using anti-TMEM119 and anti-Iba1 staining. Scale bar, 20 μm. m Quantification of cellular area following Aβo exposure, demonstrating restoration of microglial morphology by G1899. n Representative immunoblot images of anti-iNOS (activation marker), anti-TMEM119, and β-actin with quantification of o iNOS and p TMEM119 normalized to β-actin. Error bars indicate mean ± SEM. Statistical significance is determined by one-way ANOVA (c, d, f, g, i-p) or two-way ANOVA (h) with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant.

Article Snippet: Biolegend, #137001), anti- CD68 (1:1000, CST, #9778S), anti Caspase1 (1:500, Adipogen Life Sciences, AG-20B-0044- C100), anti-ASC (1:1000, Adipogen Life Sciences, AG-25B-0006), anti-Iba1 (1:1000, Wako, #019- 19741), anti-pY705 STAT3 (1:1000, CST, #9145S), anti-pS727 STAT3 (1:1000, CST, #9134S), anti-total STAT3 (1:1000, CST, #9139S), anti-Presenilin1 (1:1000, CST, #3622S), anti-APP (1:1000, MilliporeSigma, #MAB348), anti-Syntaxin 1A (1:1000, Biolegend, #827001), anti-IL-1β (1:1000, CST, #12242S), anti-Aβ (4G8) (1:1,000, Biolegend, #800708), anti-Aβ (6E10) (1:1,000, Biolegend, #803014) or anti-β-actin (1:5000, Sigma, #A3854-200UL) followed by HRP- conjugated rabbit (#32260) or mouse (#32230) secondary antibodies (1:5000, Thermo) for 1 hr at room temperature.

Techniques: Activation Assay, Western Blot, Marker, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: G1899, an American ginseng extract, alleviates neuroinflammation and cognitive impairment in models of Alzheimer’s disease

doi: 10.1101/2025.09.04.674314

Figure Lengend Snippet: Attenuation of hippocampal neuroinflammation by G1899 in 5xFAD/+ mice. a Representative immunoblot images showing expression of Aβ, IL-1β, caspase-1, pY705 STAT3, Iba1, and Syntaxin 1A in the hippocampus of WT, 5xFAD/+, and 5xFAD/+ mice treated with 300 mg/kg G1899. Quantification of relative protein expression of b Aβ (6E10), c IL-1β, d caspase-1, e pY705 STAT3, f Iba1, and g Syntaxin 1A. h Representative immunofluorescence images detecting Aβ (4G8) co-stained with Iba1 in the dorsal subiculum of the hippocampus, shown at 10× magnification for global Iba1 intensity (Scale bar, 100 μm) and i 63× magnification to visualize microglial recruitment toward Aβ plaques (Scale bar, 10 μm). Quantification of j Iba1 immunofluorescence intensity and k the number of microglia recruited toward Aβ plaques. Error bars indicate mean ± SEM. Statistical significance is determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Biolegend, #137001), anti- CD68 (1:1000, CST, #9778S), anti Caspase1 (1:500, Adipogen Life Sciences, AG-20B-0044- C100), anti-ASC (1:1000, Adipogen Life Sciences, AG-25B-0006), anti-Iba1 (1:1000, Wako, #019- 19741), anti-pY705 STAT3 (1:1000, CST, #9145S), anti-pS727 STAT3 (1:1000, CST, #9134S), anti-total STAT3 (1:1000, CST, #9139S), anti-Presenilin1 (1:1000, CST, #3622S), anti-APP (1:1000, MilliporeSigma, #MAB348), anti-Syntaxin 1A (1:1000, Biolegend, #827001), anti-IL-1β (1:1000, CST, #12242S), anti-Aβ (4G8) (1:1,000, Biolegend, #800708), anti-Aβ (6E10) (1:1,000, Biolegend, #803014) or anti-β-actin (1:5000, Sigma, #A3854-200UL) followed by HRP- conjugated rabbit (#32260) or mouse (#32230) secondary antibodies (1:5000, Thermo) for 1 hr at room temperature.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: G1899, an American ginseng extract, alleviates neuroinflammation and cognitive impairment in models of Alzheimer’s disease

doi: 10.1101/2025.09.04.674314

Figure Lengend Snippet: Suppression of inflammatory activation by G1899 in human induced microglia (iMG). a Representative immunoblot images showing expression of Oct-4a, Nanog, CD34, CD68, TMEM119, Iba1, and β-actin during stepwise stages of iMG differentiation. Quantification of b Oct-4a, c Nanog, d CD34, e CD68, f TMEM119, and g Iba1 expression across differentiation stages. h Representative immunoblot images showing expression of NLRP3, IL-1β, STAT3, pY705 STAT3, pS727 STAT3, Iba1, Aβ (6E10 and 4G8), and β-actin. Quantification of i NLRP3, j IL-1β, k STAT3, l pY705 STAT3, m pS727 STAT3, n Iba1, o Aβ (6E10), and p Aβ (4G8). Q Representative immunoblot images showing expression of TMEM119, CD68, Iba1, and β-actin, with quantification of r TMEM119 and s CD68. t Representative immunofluorescence images of iMG co-stained with Iba1 and TMEM119. Scale bar, 10 μm. u Quantification of relative cell size in . Error bars represent mean ± SEM. Statistical significance was determined by one-way or two-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant.

Article Snippet: Biolegend, #137001), anti- CD68 (1:1000, CST, #9778S), anti Caspase1 (1:500, Adipogen Life Sciences, AG-20B-0044- C100), anti-ASC (1:1000, Adipogen Life Sciences, AG-25B-0006), anti-Iba1 (1:1000, Wako, #019- 19741), anti-pY705 STAT3 (1:1000, CST, #9145S), anti-pS727 STAT3 (1:1000, CST, #9134S), anti-total STAT3 (1:1000, CST, #9139S), anti-Presenilin1 (1:1000, CST, #3622S), anti-APP (1:1000, MilliporeSigma, #MAB348), anti-Syntaxin 1A (1:1000, Biolegend, #827001), anti-IL-1β (1:1000, CST, #12242S), anti-Aβ (4G8) (1:1,000, Biolegend, #800708), anti-Aβ (6E10) (1:1,000, Biolegend, #803014) or anti-β-actin (1:5000, Sigma, #A3854-200UL) followed by HRP- conjugated rabbit (#32260) or mouse (#32230) secondary antibodies (1:5000, Thermo) for 1 hr at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Developmental cell

Article Title: An ILK/STAT3 pathway controls glioblastoma stem cell plasticity.

doi: 10.1016/j.devcel.2024.09.003

Figure Lengend Snippet: Figure 4. An ILK/STAT3 pathway directs astrocyte-like state transition (A) Differential enrichment of the indicated proteins and phosphorylation sites in NPE vs. NPEILK/ + BMP-4, determined by RPPA. Annotations indicate cell line and log10(p) values (n = 4). (B) Quantification of Smad1/5 pS463/465:Smad1 in the indicated cells (n = 4). (C) As in (B) for STAT3 pY705:STAT3. (D) As in (B) for STAT3 pS727:STAT3. (E) Expression of the indicated factors in cells ± BMP-4. (F) Quantification of (D) (n = 3). (G) Left: expression of the indicated factors in NPE cells treated with siLuc or siStat3 ± BMP-4. Right: quantification of left (n = 3). (H) Left: expression of the indicated factors in NPE cells ± BMP-4 treated with baricitinib (n = 3). Right: quantification of left (n = 3).

Article Snippet: Antibodies used for Western blotting were anti-ILK (BD Biosciences; #611803, 1:2000), anti-STAT3 (Cell Signalling Technology; #12640, 1:1000), anti-STAT3 pY705 (Cell Signalling Technology; #9145, 1:1000), anti-GFAP (Cell Signalling Technology; #3670, 1:2000), anti-PINCH (Cell Signalling Technology; #11890, 1:500), anti-ɑ-Parvin (Cell Signalling Technology; #8190, 1:1000), antiNF1 (Bethyl; #A300-140A, 1:2000), anti-PTEN (Cell Signalling Technology; #9559, 1:1000), anti-GFP (Roche; #11814460001, 1:1000), anti-EGFR (Cell Signalling Technology; #4267, 1:1000), anti-GAPDH (CST; #5174, 1:2000) and anti-ɑ-Tubulin (Cell Signalling Technology; #3873, 1:2000).

Techniques: Phospho-proteomics, Expressing

Journal: Experimental & molecular medicine

Article Title: STAT3 promotes NLRP3 inflammasome activation by mediating NLRP3 mitochondrial translocation.

doi: 10.1038/s12276-024-01298-9

Figure Lengend Snippet: Fig. 1 Identification of STAT3 inhibitors that regulate the NLRP3 inflammasome. a Screening for small molecule compounds obtained from Selleck Co. This figure template, “Screening flow chart,” was assembled using dynamic BioRender assets (icons, lines, shapes and/or text) and is fully editable. b Percentage inhibition of IL-1β secretion by each compound. The IL-1β secretion level relative to 10% of that in the control group was used as a cutoff (left), and the chemical structures of the compounds napabucasin, BP-1-102 and C188-9 used to inhibit STAT3 are shown (right).

Article Snippet: The anti-caspase-1 antibody (Abcam, ab179515), anti-IL-1β antibody (RD Systems, AF-401-NA), antiNLRP3 antibody (AdipoGen, AG-20B-0014-C100), anti-ASC antibody (AdipoGen, AG-25B-0006-C100), anti-β-actin antibody (Cell Signaling Technology, #3700), anti-STAT3 antibody (Cell Signaling Technology, #4904), anti-pY705-STAT3 antibody (Cell Signaling Technology, #9145), anti-pS727-STAT3 antibody (Cell Signaling Technology, #9134), antiGAPDH antibody (Cell Signaling Technology, #2118), anti-VDAC antibody (Cell Signaling Technology, #4866), anti-LaminA/C antibody (Cell Signaling Technology, #4777), anti-DYKDDDDK tag antibody (Cell Signaling Technology, #2368), and anti-Myc-Tag Antibody (Cell Signaling Technology, #2272) were obtained from the indicated suppliers.

Techniques: Inhibition, Control

Journal: Experimental & molecular medicine

Article Title: STAT3 promotes NLRP3 inflammasome activation by mediating NLRP3 mitochondrial translocation.

doi: 10.1038/s12276-024-01298-9

Figure Lengend Snippet: Fig. 3 STAT3 promotes NLRP3 inflammasome activation at the transcriptional level. a Quantitative PCR analysis of STAT3 mRNA expression in mouse peritoneal macrophages after transfection with NC-siRNA or STAT3-siRNA for 48 h. b IL-1β and TNF-α secretion in supernatants from mouse peritoneal macrophages transfected as described in (a) and then treated with the indicated stimuli. c, d Relative NLRP3 and IL-1β mRNA expression (c) in peritoneal macrophages treated with LPS for 2 h and 4 h or not treated after transfection as described in (a). Target mRNA expression was normalized to the expression of β-actin as the reference gene. Immunoblot analysis of NLRP3 and Pro-IL-1β expression in mouse peritoneal macrophages transfected as described; the photographs are representative of three biologically independent experiments with similar results (d). The results are presented as the means ± SDs; n = 3 biologically independent experiments (a–c). Statistical analyses were carried out via independent sample t-test in (a) or two-way ANOVA with the Bonferroni correction in (b, c). *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: The anti-caspase-1 antibody (Abcam, ab179515), anti-IL-1β antibody (RD Systems, AF-401-NA), antiNLRP3 antibody (AdipoGen, AG-20B-0014-C100), anti-ASC antibody (AdipoGen, AG-25B-0006-C100), anti-β-actin antibody (Cell Signaling Technology, #3700), anti-STAT3 antibody (Cell Signaling Technology, #4904), anti-pY705-STAT3 antibody (Cell Signaling Technology, #9145), anti-pS727-STAT3 antibody (Cell Signaling Technology, #9134), antiGAPDH antibody (Cell Signaling Technology, #2118), anti-VDAC antibody (Cell Signaling Technology, #4866), anti-LaminA/C antibody (Cell Signaling Technology, #4777), anti-DYKDDDDK tag antibody (Cell Signaling Technology, #2368), and anti-Myc-Tag Antibody (Cell Signaling Technology, #2272) were obtained from the indicated suppliers.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Transfection, Western Blot

Journal: Experimental & molecular medicine

Article Title: STAT3 promotes NLRP3 inflammasome activation by mediating NLRP3 mitochondrial translocation.

doi: 10.1038/s12276-024-01298-9

Figure Lengend Snippet: Fig. 4 Cytoplasmic STAT3 interacts with NLRP3. a Immunoblot analysis of cytoplasmic and nuclear components after treatment with the corresponding stimuli described above. b Immunoprecipitation and immunoblot analysis of lysates from HEK293T cells transfected with Flag- STAT3, Myc-NLRP3, Myc-ASC, Myc-STAT3, Flag-caspase-1, or Flag-Nek7. Immunoprecipitation was performed with anti-Flag (up) and anti-Myc (down) antibodies, and immunoblotting was performed with anti-Myc and anti-Flag antibodies, respectively. c Immunoblot analysis of lysates from mouse peritoneal macrophages treated with the indicated stimuli. IP was performed with an anti-STAT3 antibody, and immunoblotting was then performed with an anti-NLRP3 antibody. d The physical interaction between NLRP3 and STAT3 was visualized as red puncta by a PLA in mouse peritoneal macrophages primed with 100 ng/mL LPS for 3 h and then stimulated with nigericin (10 μM) or ATP (5 mM) for 1 h. Scale bar: 20 μm. e Myc-tagged NLRP3 or its mutants and Flag-STAT3 were cotransfected into HEK293T cells for 24 h prior to immunoprecipitation with anti-Myc beads and western blotting (bottom). Schematic diagram of NLRP3 and its truncation mutants (top). f Myc-STAT3 or its mutants and Flag-NLRP3 were cotransfected into HEK293T cells for 24 h prior to immunoprecipitation with anti-Myc beads and western blotting (bottom). Schematic diagram of STAT3 and its truncation mutants (top). The results are presented as the means ± SDs, and representative photographs of three biologically independent experiments with similar results are shown.

Article Snippet: The anti-caspase-1 antibody (Abcam, ab179515), anti-IL-1β antibody (RD Systems, AF-401-NA), antiNLRP3 antibody (AdipoGen, AG-20B-0014-C100), anti-ASC antibody (AdipoGen, AG-25B-0006-C100), anti-β-actin antibody (Cell Signaling Technology, #3700), anti-STAT3 antibody (Cell Signaling Technology, #4904), anti-pY705-STAT3 antibody (Cell Signaling Technology, #9145), anti-pS727-STAT3 antibody (Cell Signaling Technology, #9134), antiGAPDH antibody (Cell Signaling Technology, #2118), anti-VDAC antibody (Cell Signaling Technology, #4866), anti-LaminA/C antibody (Cell Signaling Technology, #4777), anti-DYKDDDDK tag antibody (Cell Signaling Technology, #2368), and anti-Myc-Tag Antibody (Cell Signaling Technology, #2272) were obtained from the indicated suppliers.

Techniques: Western Blot, Immunoprecipitation, Transfection

Journal: Experimental & molecular medicine

Article Title: STAT3 promotes NLRP3 inflammasome activation by mediating NLRP3 mitochondrial translocation.

doi: 10.1038/s12276-024-01298-9

Figure Lengend Snippet: Fig. 6 Targeting STAT3 alleviates NLRP3-associated inflammation. a, b Wild-type C57BL/6 mice were administered DMSO or napabucasin (5 mg/kg) via intraperitoneal (i.p.) injection 30 min before i.p. injection of MSU (2 mg per mouse) (n = 5 biologically independent mice) for 6 h. Quantification of neutrophils (a) and ELISA of IL-1β secretion (b) in the peritoneal lavage fluid. c–e Wild-type C57BL/6 mice were administered 20 mg/kg LPS via intraperitoneal (i.p.) injection (n = 5 biologically independent mice) for 8 h with or without napabucasin (5 mg/kg). ELISA results showing the serum concentrations of IL-1β (c) and IL-6 (d) and images of H&E-stained lung tissue sections (e). Scale bar: 50 μm. The data are representative of three independent experiments. The results are presented as the means ± SEMs. Statistical analyses were carried out via two-way ANOVA in (a–d). *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: The anti-caspase-1 antibody (Abcam, ab179515), anti-IL-1β antibody (RD Systems, AF-401-NA), antiNLRP3 antibody (AdipoGen, AG-20B-0014-C100), anti-ASC antibody (AdipoGen, AG-25B-0006-C100), anti-β-actin antibody (Cell Signaling Technology, #3700), anti-STAT3 antibody (Cell Signaling Technology, #4904), anti-pY705-STAT3 antibody (Cell Signaling Technology, #9145), anti-pS727-STAT3 antibody (Cell Signaling Technology, #9134), antiGAPDH antibody (Cell Signaling Technology, #2118), anti-VDAC antibody (Cell Signaling Technology, #4866), anti-LaminA/C antibody (Cell Signaling Technology, #4777), anti-DYKDDDDK tag antibody (Cell Signaling Technology, #2368), and anti-Myc-Tag Antibody (Cell Signaling Technology, #2272) were obtained from the indicated suppliers.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Experimental & molecular medicine

Article Title: STAT3 promotes NLRP3 inflammasome activation by mediating NLRP3 mitochondrial translocation.

doi: 10.1038/s12276-024-01298-9

Figure Lengend Snippet: Fig. 7 Model showing the role of STAT3 in NLRP3 inflammasome activation. STAT3 promotes NLRP3 inflammasome activation by mediating NLRP3 mitochondrial translocation, and this process is inhibited by napabucasin through disruption of the interaction between STAT3 and NLRP3.

Article Snippet: The anti-caspase-1 antibody (Abcam, ab179515), anti-IL-1β antibody (RD Systems, AF-401-NA), antiNLRP3 antibody (AdipoGen, AG-20B-0014-C100), anti-ASC antibody (AdipoGen, AG-25B-0006-C100), anti-β-actin antibody (Cell Signaling Technology, #3700), anti-STAT3 antibody (Cell Signaling Technology, #4904), anti-pY705-STAT3 antibody (Cell Signaling Technology, #9145), anti-pS727-STAT3 antibody (Cell Signaling Technology, #9134), antiGAPDH antibody (Cell Signaling Technology, #2118), anti-VDAC antibody (Cell Signaling Technology, #4866), anti-LaminA/C antibody (Cell Signaling Technology, #4777), anti-DYKDDDDK tag antibody (Cell Signaling Technology, #2368), and anti-Myc-Tag Antibody (Cell Signaling Technology, #2272) were obtained from the indicated suppliers.

Techniques: Activation Assay, Translocation Assay, Disruption