phosphorylated ser727  (Cell Signaling Technology Inc)

Journal: Frontiers in Pharmacology

Article Title: Ruxolitinib alleviates DSS-induced acute ulcerative colitis by inhibiting STAT1 phosphorylation and reducing MDSC infiltration

doi: 10.3389/fphar.2025.1572534

Figure Lengend Snippet: Effect of ruxolitinib on JAK/STAT signaling pathway-related genes and inhibition of interferon (IFN)-γ-Induced STAT1 activation by ruxolitinib. (A) Heatmap of transcription levels (fold-changes) of the JAK/STAT signaling-related genes in CCD841 and Jurkat cells after treatment with IFN-γ and ruxolitinib. (B) Nuclear extracts prepared from CCD841 cells with or without IFN-γ activation and analyzed for STAT1 activation using electrophoretic mobility shift assay (EMSA) with the STAT1-binding consensus sequence DNA probe. Ruxolitinib at 50, 250, and 500 nM was incubated with STAT1-DNA complexes. RUX: ruxolitinib.

Article Snippet: DSS was purchased from MP Biomedicals; Ruxolitinib was purchased from Selleck; The following antibodies were used: APC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 (BioLegend), STAT1 and phosphorylated STAT1 (Tyr701) (Proteintech), phosphorylated STAT1 (Ser727) (Cell Signaling Technology).

Techniques: Inhibition, Activation Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Incubation

Journal: Frontiers in Pharmacology

Article Title: Ruxolitinib alleviates DSS-induced acute ulcerative colitis by inhibiting STAT1 phosphorylation and reducing MDSC infiltration

doi: 10.3389/fphar.2025.1572534

Figure Lengend Snippet: Inhibition of STAT1 phosphorylation by ruxolitinib in immune cells and normal intestinal epithelial cells. (A) Western blot analysis of p-STAT1Y701, p-STAT1S727, STAT1, and β-actin in Jurkat, RAW and CCD841 cells treated with 50 IU/mL IFN-γ for the indicated times. (B–D) Quantitative analysis of p-STAT1Y701, p-STAT1S727, STAT1 levels in (B) Jurkat, (C) RAW, and (D) CCD841 cells from (A) . (E) Western blot analysis of p-STAT1Y701, p-STAT1S727, STAT1, and β-actin in cells pretreated with IFN-γ (8 h) followed by Ruxolitinib (RUX, 24 h) at indicated concentrations (μM). (F–H) Quantitative analysis of p-STAT1Y701, p-STAT1S727, STAT1 levels in (F) Jurkat, (G) RAW, and (H) CCD841 cells from (E) . # P < 0.05, ## P < 0.01, ### P < 0.001 vs. 0 μM RUX (IFN-γ-treated). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, * P < 0.001 vs. the group with IFN-γ concentration at 0; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the IFN-γ-treated group with RUX concentration at 0. RUX: ruxolitinib.

Article Snippet: DSS was purchased from MP Biomedicals; Ruxolitinib was purchased from Selleck; The following antibodies were used: APC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 (BioLegend), STAT1 and phosphorylated STAT1 (Tyr701) (Proteintech), phosphorylated STAT1 (Ser727) (Cell Signaling Technology).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Concentration Assay

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 5. Effect of IL-1b, glucocorticoid, and IFN-c on STAT1 phosphorylation. A549 cells were either untreated or treated with IFN-c (10 ng/ml) prior to harvesting for western blot analysis at the indicated times. Representative blots of (A) phospho-STAT1 (P-STAT1 Y701) and GAPDH and (B) P-STAT1 (S727), total STAT1, and GAPDH from N independent experiments are shown. In each case, densitometric data normalized to GAPDH are plotted as means ± S.D. (error bars). Significance relative to untreated at 15 minutes was assessed by ANOVA with a Dunnett’s mul- tiple comparison test. (C) A549 cells were either not treated or treated with IL-1b (1 ng/ml), IFN-c (10 ng/ml), and/or Dex (1 mM) as indicated. Cells were harvested at 1 hour for western blot analysis of P-STAT1 (Y701 and S727) and GAPDH. Representative blots of P-STAT1 (Y701 & S727) and GAPDH from N independent experiments are shown. Densitometric data for P-STAT1 (Y701) (upper graph) and P-STAT1 (S727) (lower graph) were normalized to GAPDH, expressed as means ± S.D., and plotted as bar graphs overlaid with scatter plots. Significance relative to un- treated (*) or IL-1b1IFN-c (#) was tested using ANOVA with a Tukey post hoc test. *, #P # 0.05; **, ##P # 0.01; ***, ###P # 0.001. Scatterplots for all data in (A) and (B) are shown as Supplemental Fig. 6.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Phospho-proteomics, Western Blot, Comparison

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 6. Effect of JAK inhibitors on STAT1 phosphorylation. (A) A549 cells were pretreated with AZD1480, barcitinib and ruxolitinib at the indi- cated concentrations for 30 minutes, followed by stimulation with IFN-c (10 ng/ml) for 1 hour. Cells were harvested for western blot analysis of phospho-STAT1 (P-STAT1) and GAPDH. Representative blots for P-STAT1 (Y701), P-STAT1 (S727), and GAPDH from five independent experi- ments are shown. (B) Densitometric data for Y701 P-STAT1 and S727 P-STAT1 from western blots shown in (A) were normalized to GAPDH and plotted as mean ± S.D. (C) Data were expressed as percent IFN-c effect for each of AZD1480, barcitinib, and ruxolitinib and plotted as means ± S.D. Log EC50 of each compound for the inhibition of P-STAT1 (Y701) and P-STAT1 (S727) is expressed as mean ± S.D. and shown as scatter plots in the inserts within each graph of (C). Note: one dataset was excluded for ruxolitinib, and the full data are shown as Supplemental Fig. 8. (D and E) A549 cells were either not treated or pretreated with AZD1480, barcitinib, and ruxolitinib, each at 1 mM, for 1 hour prior to stimulation with IL-1b (1 ng/ml) and IFN-c (10 ng/ml) as indicated. Cells were harvested at 1 hour for western blot analysis of P-STAT1 and GAPDH. Representative blots for P-STAT1 (Y701 & S727) and GAPDH from N independent experiments are shown. Densitometric data for (D) P-STAT (S727) and (E) P-STAT1 (Y701) were normalized to GAPDH, expressed as means ± S.D., and plotted as bar graphs overlaid with scatter plots. Significance relative to untreated (*), IFN-c (#), or IL-1b 1 IFN-c ($) was tested using ANOVA with a Tukey post hoc test. *, #, $P # 0.05; **, ##, $$P # 0.01; ***, ###, $$$P # 0.001.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Phospho-proteomics, Western Blot, Inhibition

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 7. Characterization of a STAT1-driven luciferase reporter. (A) A549 cells stably transfected with a luciferase reporter containing three copies of a consensus STAT1-binding motif [shown in (B)] were either not stimulated (NS) or stimulated with the indicated concentrations of IFN-c prior to harvesting for luciferase assay after 6 hours. Luciferase activity from N independent experiments was expressed as fold of NS and plotted as mean ± S.D. (B) (Upper panel) reporter construct showing three copies of the consensus/wild-type (WT; bold blue) or mutant (Mut; bold blue 1 red) STAT1-binding motif is shown. KpnI and XhoI restriction sites used to clone the construct in pGL3.TATA.neo vector are underlined, with cut sites shown as black arrowheads. (Lower panel) A549 cells were stably transfected with the WT or Mut STAT1 reporter constructs and either NS or treated with IFN-c (10 ng/ml) for 6 hours followed by harvesting for luciferase assay. Luciferase activity from N independent experiments was expressed as fold of NS, and mean ± S.D. was plotted as bar graphs overlaid with scatter plots. Significance relative to untreated cells was deter- mined using paired t test. (C) A549 cells were either NS or incubated with lipid control (lip) alone or with the indicated concentrations of either control siRNA pool (C si-pool), or STAT1 siRNA pool (STAT1 si-pool) for 48 hours. Cells were then incubated overnight with serum-free media prior to harvesting for western blot analysis. Representative blots of STAT1 and GAPDH from N independent experiments are shown. Data ob- tained from densitometry analysis were normalized to GAPDH, and mean ± S.D. was plotted as bar graphs overlaid with scatter plots. Signifi- cance relative to C si-pool at each siRNA concentration was tested by paired t test. *P # 0.05; **P # 0.01; ***P # 0.001. (D) A549 cells stably transfected with the WT STAT1 reporter construct were treated with lipid or siRNAs as described in (C) prior to stimulation with IFN-c (10 ng/ml) for 6 hours. Cells were harvested for luciferase assay, and raw luciferase units (RLU) from N independent experiments were expressed as mean ± S.D. and plotted as bar graphs overlaid with scatter plots. Significance relative to C si-pool at each siRNA concentration was tested by paired t test. (E) A549 cells stably transfected with the WT STAT1 reporter construct were either NS or pretreated with the indicated concentra- tions of AZD1480, barcitinib, or ruxolitinib for 30 minutes followed by stimulation with IFN-c (10 ng/ml). Cells were harvested for luciferase assay after 6 hours. Luciferase activity from N independent experiments was expressed as fold of NS and plotted as mean ± S.D. Scatterplots for all data in (A) and e are shown as Supplemental Fig. 9.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Luciferase, Stable Transfection, Transfection, Binding Assay, Activity Assay, Construct, Mutagenesis, Plasmid Preparation, Incubation, Control, Western Blot, Concentration Assay

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 9. Effect of p65, GR, and STAT1 knockdown on IL-1b, IFN-c, and dexamethasone-induced TLR2 expression in A549 cells. (A) A549 cells were incubated with pools of either control siRNAs (C si-pool), p65 siRNAs (p65 si-pool), GR siRNAs (GR si-pool), or STAT1 siRNAs (STAT1 si-pool) (1 nM each) for 48 hours. Cells were then incubated overnight with serum-free media prior to harvesting for western blot analysis. Representative blots of p65, GR, STAT1, and GAPDH from five independent experiments are shown. (B–E) A549 cells were incubated with siRNA pools as in (A), followed by treatment with IL-1b (1 ng/ml), IFN-c (10 ng/ml), and/or Dex (1 mM), as indicated, for 24 hours. Cells were harvested for western blot analysis. Representative blots of TLR2 and GAPDH from N independent experiments are shown. Densitometric data were normalized to GAPDH, expressed as fold of untreated, and mean ± S.D. were plotted as bar graphs overlaid with scatter plots. Significance relative to C si-pool in each case was tested using ANOVA with a Dunnett’s post hoc test. *P # 0.05; **P # 0.01; ***P # 0.001.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Knockdown, Expressing, Incubation, Control, Western Blot

Journal: Oncology Reports

Article Title: GBP2 promotes clear cell renal cell carcinoma progression through immune infiltration and regulation of PD‑L1 expression via STAT1 signaling

doi: 10.3892/or.2023.8486

Figure Lengend Snippet: Correlation analysis between guanylate-binding protein 2 and markers of immune cells in Tumor Immune Estimation Resource 2.0 database.

Article Snippet: The NC membranes were blocked in 5% skimmed milk for 1 h at 20–25°C and were incubated for 8–12 h at 4°C with specific primary antibodies against STAT1 (1:1,000; cat. no. R25799), phosphorylated (p)-STAT1 (Ser727) (1:1,000; cat. no. R25797; both from Zen BioScience), PD-L1 (1:5,000; cat. no. 381830), β-actin (1:5,000; cat. no. 66009-1-lg), Flag (1:6,000; cat. no. 20543-1-AP) and GBP2 (1:1,000; cat. no. 27299-1-AP; all from Proteintech Group, Inc.).

Techniques:

Journal: Oncology Reports

Article Title: GBP2 promotes clear cell renal cell carcinoma progression through immune infiltration and regulation of PD‑L1 expression via STAT1 signaling

doi: 10.3892/or.2023.8486

Figure Lengend Snippet: GBP2 regulates PD-L1 expression by interacting with STAT1. (A) Protein levels of GBP2, STAT1 and p-STAT1 (Ser 727) were examined by western blotting with shNS and shGBP2#1 in Caki-1 and 786-O cell lines. (B) Protein expression of GBP2, STAT1 and p-STAT1 (Ser 727) were examined by western blotting with vector and GBP2 OE in Caki-1 and 786-O cells. (C) Caki-1 and 786-O cells were treated with DMSO or Fludarabine (50 µM) for 36 h prior examining PD-L1 mRNA levels by reverse transcription-quantitative PCR. (D) Caki-1 and 786-O cells were treated with DMSO or Fludarabine (50 µM) for 36 h prior to immunoblot analysis of PD-L1 and GBP2 levels. (E) Concentrations of PD-L1, STAT1 and p-STAT1 were examined by western blotting in Caki-1 and 786-O (vector, GBP2 OE and GBP2 OE cells transfected with siNC or siSTAT1#1 and siSTAT1#2). (F) The protein-protein interaction between GBP2 and STAT1 was validated using immunoprecipitations followed by western blot analyses with indicated antibodies in Caki-1 cells and 786-O cells. IgG was used as an immunoprecipitation control. (G) A possible mechanism for GBP2-promoted PD-L1 upregulation and immunosuppression in clear cell renal cell carcinoma. Data are expressed as the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. GBP 2, guanylate-binding protein 2; PD-L1, programmed death-ligand 1; p-, phosphorylated; sh-, short hairpin; OE, overexpression; si-, small interfering; NC, negative control; n.s., not significant.

Article Snippet: The NC membranes were blocked in 5% skimmed milk for 1 h at 20–25°C and were incubated for 8–12 h at 4°C with specific primary antibodies against STAT1 (1:1,000; cat. no. R25799), phosphorylated (p)-STAT1 (Ser727) (1:1,000; cat. no. R25797; both from Zen BioScience), PD-L1 (1:5,000; cat. no. 381830), β-actin (1:5,000; cat. no. 66009-1-lg), Flag (1:6,000; cat. no. 20543-1-AP) and GBP2 (1:1,000; cat. no. 27299-1-AP; all from Proteintech Group, Inc.).

Techniques: Expressing, Western Blot, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Immunoprecipitation, Control, Binding Assay, Over Expression, Negative Control