Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: mPGES1-dependent PGE 2 controls antigen-specific Th17 and Th1 responses by regulating T autocrine and paracrine PGE 2 production

doi: 10.4049/jimmunol.1601808

Figure Lengend Snippet: (A) WT or mPGES1−/− (KO) freshly isolated naïve CD4+ cells were analyzed during Th17 polarization. Levels of rorc mRNA expression the absence or presence of 10 nM PGE2 at day 2 are shown on the bar graphs, while RORγt and IL-17A intracellular expression at day 4 are shown in representative the dot plots. The far right bar graph summarizes the double positive RORγt+IL-17A+ percentages in 4 replicate experiments, each started with a pool of naïve CD4+ cells from 3 mice. (B) The phosphorylation status of STAT3 (Y705) and STAT5 (Y694) within RORγt+ cells was also examined in presence (shaded histograms) or absence (open histograms) of PGE2. (C) Cells undergoing Th1 differentiation were analyzed for their mRNA levels of expression of tbx21 at day 2, and their intracellular expression of Tbet and IFNγ at day 4 (representative dot plots), with the right bar graph summarizing Tbet+IFNγ+ percentages in 4 replicates. (D) mRNA levels of ptgs2 under Th1 or Th17 polarization at day 2 in presence or absence of PGE2. All values are relative to WT Th17 cells, and ** indicates a P value <0.01 using a 2-tailed heteroscedastic Student’s T-test.

Article Snippet: The staining was performed according to manufacturer’s instructions for those antibodies, and for phosphoflow antibodies from BD Pharmingen clones 4/P Stat3 (Y705) and 4/7 Stat5 (Y694), using Perm Buffer III.

Techniques: Isolation, Expressing

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Upregulation of IL-9 and inflammatory cytokines as well as the activation of Notch1 signaling during EAE process. a The level of IL-9 in spinal cords of EAE mice with different clinical scores was measured by real-time PCR and Western blot assay, respectively. b The expressions of GFAP, NICD and p-STAT3 in spinal cords along with EAE process were detected by Western blot assay. c The mRNA levels of IL-6, TNF-α, IP-10, and MCP-1 in spinal cords were determined using real-time PCR. d The secretion levels of IL-6, TNF-α, IP-10 and MCP-1 in the sera were detected by Cytometric Bead Array (CBA) during the EAE process. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus PBS group ( n = 6/group, one-way ANOVA). These results were repeated four times. Data are represented as the mean ± SEM

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

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

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: IL-9 activates the Notch1 pathway and promotes inflammatory cytokines production in astrocytes. Primary mouse astrocytes were incubated in a serum-free medium overnight followed by treating with IL-9 at the indicated time point. a The expression changes of GFAP, NICD, and p-STAT3 were analyzed by Western blot assay. b Immunofluorescent staining for GFAP (green), Notch1 (red), and nuclear staining of DAPI (blue) in cultured astrocytes with IL-9 treatment for 6 h. Scale bars, 50 μm. c The mRNA levels of IL-6, TNF-α, IP-10, and MCP-1 in astrocytes were detected using real-time PCR assay. d The secretion levels of IL-6, TNF-α, IP-10, and MCP-1 in the supernatant of astrocytes were measured by CBA assay. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus DMEM group (one-way ANOVA). The data are from three independent experiments and represented as the mean ± SEM

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Incubation, Expressing, Western Blot, Staining, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Inhibition of Gm13568 downregulates Notch1 signaling activation as well as inflammatory cytokine production in astrocytes by IL-9. a Primary mouse astrocytes were infected with recombinant lentivirus, LV-Inhibit-Gm13568 and LV-ctrl, for 72 h, respectively. Then, the astrocytes were incubated in a serum-free medium overnight followed by IL-9 stimulation for 6 h. Western blot assay was used for measuring the protein expressions of GFAP, Notch1/NICD, and p-STAT3. b The mRNA levels of IL-6, TNF-α and IP-10 in astrocytes were measured by real-time PCR assay. c The secretion of IL-6, TNF-α, and IP-10 in the supernatant of astrocytes were analyzed by CBA assay. The data are represented as the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus DMEM group; ## p < 0.01 and ### p < 0.001 versus LV-ctrl+IL-9 group (one-way ANOVA, the two-tailed Student’s t test)

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Inhibition, Activation Assay, Infection, Recombinant, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Knockdown of Notch1 in astrocytes suppresses inflammation and alleviates EAE in mice. Mice were subjected to recombinant lentiviruses, LV-ctrl or LV-Notch1-shRNA, for 7 days, followed by MOG 35-55 immunization for 23 days ( n = 10 mice per group). a The clinical scores of EAE mice with LV-ctrl and LV-Notch1-shRNA . b The expressions of IL-9, GFAP, NICD, and p-STAT3 in the spinal cords were detected using western blot assay. c , d The changes of IL-6, TNF-α, and IP-10 in the spinal cords and peripheral blood of the LV-ctrl and LV-Notch1-shRNA mice were evaluated by real-time PCR and CBA assay, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. PBS group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus LV-ctrl group (one-way ANOVA, the two-tailed Student’s t test). Results are represented as mean ± SEM. e The infiltration of inflammatory cells in spinal cords was investigated using hematoxylin and eosin (H&E) staining (scale bars, 50 μm). f The medullary sheath damages from spinal cords were observed via luxol fast blue (LFB) staining (scale bars, 50 μm). Boxed areas in the upper rows are presented enlarged underneath

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Knockdown, Recombinant, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test, Staining

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: Downregulation of BAG3 affects p-STAT3(705) and p-STAT3(727)

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 regulates p-STAT3(705) and p-STAT3(727) through p-JAK2 and p-ERK1/2 respectively to promote VSMCs migration

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques: Migration

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 modulates STAT3 phosphorylation by regulating the interaction of STAT3 with JAK2 and ERK1/2

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Bioresources and Bioprocessing

Article Title: Propionate-engineered probiotics reduce radiation-induced intestinal damage

doi: 10.1186/s40643-026-01020-9

Figure Lengend Snippet: Engineered probiotic PG mitigated radiation-induced intestinal injury by regulating the SOCS1/JAK2/STAT3 pathway. a Principal Component Analysis (PCA) revealed differences in gene expression levels among groups. b KEGG Enrichment BarPlot for IR vs. IR + PG displayed the number of differential genes in the pathway represented on the horizontal axis, pathway names on the vertical axis, and colors representing KEGG Level 1 classification. c KEGG-enriched bar graph displaying the top 20 KEGG pathways with the smallest P-values. d The results of the KEGG enrichment analysis werw presented as a scatter plot. (e) The heatmap illustrated the relative expression levels of JAK-STAT signaling pathway-related genes in the IR and IR + PG groups. f Western blot analysis demonstrated the protein expression levels of SOCS1, p-JAK2, JAK2, p-STAT3, and STAT3 across groups

Article Snippet: The primary antibodies included anti-SOCS1 (PSH09-68), JAK2 (SY0245), p-JAK2 (SY24-03), STAT3 (SY24-08), p-STAT3 (SZ43-01), and GAPDH (SA30-01), all purchased from HuaBio-Antibodies Co., Ltd (Hangzhou, China).

Techniques: Gene Expression, Expressing, Western Blot

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Changes in phosphorylation (activation) at the regulatory sites Y705 and S727 of STAT3 in mouse HL-1 cardiomyocytes following 10 min of incubation with different urocortin (Ucn) concentrations (1 and 10 NM). ( A ) Ucn induced STAT3 phosphorylation at both Y705 and S727 sites, while the expression of total STAT3 protein did not significantly vary. ( B ) Corresponding clustered column graph of the Western blot shown in Figure 1A. Data are expressed as% of control. * p<0.05, ** P<0.01

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Activation Assay, Incubation, Expressing, Western Blot, Control

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Time-dependent effects of Ucn on STAT3 phosphorylation in HL-1 cells. ( A ) Representative blots of the time-dependent effects of Ucn [10 nM] using anti-pSTAT3 (Y705 and S727) and total anti-STAT3 polyclonal antibodies. Phosphorylation of STAT3 at both Y705 and S727 sites was significantly enhanced after 15 min. However, while STAT3 phosphorylation at Y705 site further increased at 30, 60, and 120 min, STAT3 phosphorylation at S727 site peaked at 15 min, declined after 30 min, and remained stable after 1 and 2 h. The expression of total STAT3 protein was unaffected by incubation of HL-1 cells with Ucn. ( B ) Corresponding clustered column graph of the Western blot presented in . Data are expressed as% of control. * p<0.05, ** p<0.01. (C, E ) Dose-dependent effects of Ucn (ranging from 10 −11 to 10 −6 M and from 10 −12 to 10 −6 M, respectively) after 15 min of incubation using anti-pSTAT1 (Y701), total anti-STAT1, anti-pSTAT3 (Y705), anti-pSTAT3 (S727), and total anti-STAT3 polyclonal antibodies. GAPDH blot is shown as a loading control. Ucn concentrations as low as 10 −11 M were sufficient to phosphorylate STAT3 at both Y705 and S727 residues. STAT1 (Y701) phosphorylation was unaffected at any Ucn concentration. Likewise, the expression levels of total STAT1 and STAT3 proteins were unchanged. ( D, F ) Corresponding clustered column graphs of the Western blots depicted in Figure 2C and 2D, respectively. Data are expressed as% of control. * p<0.05, ** P<0.01.

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Expressing, Incubation, Western Blot, Control, Concentration Assay

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Electrophoretic mobility shift assay (EMSA) and super-shift assay of STAT3 of 5 μg nuclear extract of each sample. Ucn significantly increased STAT3 binding to DNA probe (lane C). In addition, the Ucn-induced DNA-STAT3 complex could be partially super-shifted by pre-incubation with anti-STAT3 antibody before EMSA (lane D ). Lane A shows free DNA probe only. Lane B shows the nuclear extracts from untreated HL-1 cells (Con – Control). Lane C shows nuclear extracts from HL-1 cells treated with Ucn (10 nM, for 30 min). Lane D shows nuclear extracts from HL-1 cells treated with Ucn (10 nM, for 30 min) and incubated with an anti-STAT3 antibody before EMSA. See Results section for further description

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Electrophoretic Mobility Shift Assay, Super-Shift Assay, Binding Assay, Incubation, Control

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Ucn induced IL-6 release and IL-6-mediated phosphorylation/nuclear translocation of STAT3 (Y705). ( A ) ELISA test showing the temporal release of IL-6 in the culture medium of HL-1 cells treated with Ucn 10 nM for 30 min. IL-6 release started after 1 h, peaked at 12 h, and declined thereafter, although remaining detectable at 24 h. Data are presented as the average of 3 independent experiments. ( B ) A TransAM STAT3 assay was used in HL-1 nuclear extracts to detect and quantify nuclear translocation of p-STAT3. Incubation of HL-1 cells with Ucn (10 nM) increased phosphorylation and nuclear translocation of STAT3 (Y705) in a time-dependent manner. Nuclear extracts from HepG2 cells treated with IL-6 (100 ng/ml) were used as positive control. Use of an anti-IL-6 antibody to neutralize the effects of Ucn-induced IL6 release decreased significantly STAT3 phosphorylation at Y705. * p<0.05, ** p<0.01. ( C ) A representative blot probing pSTAT3 (Y705) in the nuclear extracts from HL-1 cells tested in the TransAM assay is shown. ( D ) Western blot analysis of whole lysates of HL-1 cells treated with Ucn (10 nM) for increasing incubation times (5, 15, and 30 min) confirmed that neutralization of secreted IL-6 (using an anti-IL-6 antibody) was sufficient to block STAT3 phosphorylation at Y705, which peaked at 30 min. Overall expression of total STAT3 was unaffected. * p<0.05, ** p<0.01. ( E ) Corresponding clustered column graph of the Western blot shown in Figure 4D. Data are expressed as% of control. * p<0.05, ** P<0.01.

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Translocation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Western Blot, Neutralization, Blocking Assay, Expressing, Control

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Ucn-induced phosphorylation of Y705 and S727 residues of STAT3 via JAK and MAPK pathways. ( A ) Western blot analysis showing the inhibitory effects of AG490 (JAK2/JAK3 inhibitor) and PD98059 (MAPK kinase inhibitor) on Ucn-induced phosphorylation of Y705 and S727 residues of STAT3. HL-1 cells were incubated with Ucn (used at a concentration of 10 nM) for 30 min. The cell lysate from HL-1 cells treated with leukemia inhibitory factor (LIF, 5 ng/ml) for 15 min served as a positive control. ( B ) Corresponding clustered column graph of the Western blot shown in Figure 6A. Data are expressed as% of control. * p<0.05, ** P<0.01 ( C ) The inhibitory effects of PD98059 and AG490 on Ucn-induced activation of ERK1/2 are depicted by Western blot analysis. HL-1 cells were treated with Ucn 10 nM for 30 min. A blot of GAPDH is shown as a loading control. ( D ) Corresponding clustered column graph of the Western blot shown in Figure 6C. Data are expressed as% of control. * p<0.05, ** P<0.01. ( E ) Western blot of time course study showing the inhibitory effect of P6 (JAK inhibitor) on JAK2-mediated phosphorylation of STAT3 (Y705 residue) in HL-1 cells treated with Ucn (10 nM) for 15 and 30 min. ( F ) Corresponding clustered column graph of the Western blot presented in Figure 6E. Data are expressed as% of control. * p<0.05, ** P<0.01.

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Incubation, Concentration Assay, Positive Control, Control, Activation Assay, Residue

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: ERK1/2 and Src activate STAT3 through phosphorylation of distinct residues. ( A ) Western blot showing that Ucn activates Src and ERK1/2 in a dose-dependent manner in HL-1 cells. The expression of total Src and total ERK1/2 remained unchanged. ( B ) Corresponding clustered column graph of the Western blot presented in Figure 7A. Data are expressed as% of control. * p<0.05, ** P<0.01. ( C ) Western blot showing the dose-dependent inhibitory effects of PP2 (a specific Src family kinase inhibitor) on Ucn-induced phosphorylation of STAT3 (Y705 residue). PP2 also strongly induced the expression of total STAT3. ( D ) Corresponding clustered column graph of the Western blot depicted in Figure 7C. Data are expressed as% of control. * p<0.05, ** P<0.01. ( E ) Western blot of SYF cells incubated with increasing concentrations of Ucn. Cell lysates were prepared for Western blot analysis using anti-pSTAT3 (Y705 and S727), anti-pERK1/2, anti-STAT3, and anti-ERK1/2 antibody. ( F ) Corresponding clustered column graph of the Western blot presented in Figure 7E. Data are expressed as% of control. * p<0.05, ** P<0.01.

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Residue, Incubation

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Schematic picture summarizes the effects of Ucn-induced activation of STAT3 via JAK/ERK/Src signal cross-talk.

Article Snippet: The rabbit polyclonal anti-P-STAT1 (Y701), anti-P-STAT3 (Y705 and S727), anti-STAT1, anti-STAT3 antibodies, and a rabbit monoclonal anti-P-STAT3 (Y705) antibody were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay

Journal: Oncotarget

Article Title: Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival

doi: 10.18632/oncotarget.23422

Figure Lengend Snippet: STAT3 phosphorylation at either Tyr705 (p-STAT3 Y705) or Ser727 (p-STAT3 S727) and total STAT3 levels were detected in cell extracts from HK-2 and 786-O cells lines silenced for either CK2α or CK2β subunits by western blot using specific antibodies. The plots indicate the mean values of the densitometries of the p-STAT3 bands relative to the total STAT3 levels obtained in four different experiments. Data are represented as mean ± SEM. Values were analyzed by Student’s t -test. ( * ) and ( **** ) denote p < 0.05 and p < 0.0001, respectively.

Article Snippet: Primary antibodies used were: anti-CK2α H-286 (sc-9030, Santa Cruz Biotechnology), anti-CK2α 1AD9 (05-1431, Millipore), anti-CK2α’ (A300-199A, Bethyl Laboratories), anti-CK2β 6D5 (sc-12739, Santa Cruz Biotechnology), anti-CK2β(h) (raised in rabbits immunized with whole human recombinant CK2β as described in [ ]), anti-p-Akt1 Ser129 (ab133458, EPR6150, Abcam), anti-Akt1 (2938, C73H10, Cell Signaling), Anti-Akt2 (3063, D6G4, Cell Signaling), anti-E-Cadherin (610181, BD Biosciences), anti-Snail1 (3895, L70G2, Cell Signaling), anti-Vimentin (V6389 Sigma-Aldrich), anti-p-STAT3 Tyr705 (4113, M9C6, Cell Signaling), anti-p-STAT3 Ser727(9134, Cell Signaling), anti-STAT3 (9139, 124H6, Cell Signaling), anti-HIF-1α (ab82832, Abcam), anti-HIF-2α (ab199, Abcam), anti-β-actin (sc-47778, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot

Journal: Oncotarget

Article Title: Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival

doi: 10.18632/oncotarget.23422

Figure Lengend Snippet: ( A ) Patients exhibiting p-STAT3 S727 levels <100 and >100, correlating with good and bad prognosis, respectively , were subgrouped according to their CK2βcyt levels were above or below 41 (median value). Metastatic patients have been excluded. ( B ) COMB1 corresponds to patients exhibiting p-STAT3 S727 levels <100 and CK2βcyt >41. COMB2 includes the three other groups. No significant differences were observed among groups in COMB2. Statistical significant differences were observed between COMB1 and COMB2. ( C , D ) Kaplan-Meier estimates of 120-months overall survival according to COMB1 or COMB 2 for Low Risk (C) and High Risk (D) patients. Statistical significant differences were observed between COMB1 and COMB2 in the High Risk group.

Article Snippet: Primary antibodies used were: anti-CK2α H-286 (sc-9030, Santa Cruz Biotechnology), anti-CK2α 1AD9 (05-1431, Millipore), anti-CK2α’ (A300-199A, Bethyl Laboratories), anti-CK2β 6D5 (sc-12739, Santa Cruz Biotechnology), anti-CK2β(h) (raised in rabbits immunized with whole human recombinant CK2β as described in [ ]), anti-p-Akt1 Ser129 (ab133458, EPR6150, Abcam), anti-Akt1 (2938, C73H10, Cell Signaling), Anti-Akt2 (3063, D6G4, Cell Signaling), anti-E-Cadherin (610181, BD Biosciences), anti-Snail1 (3895, L70G2, Cell Signaling), anti-Vimentin (V6389 Sigma-Aldrich), anti-p-STAT3 Tyr705 (4113, M9C6, Cell Signaling), anti-p-STAT3 Ser727(9134, Cell Signaling), anti-STAT3 (9139, 124H6, Cell Signaling), anti-HIF-1α (ab82832, Abcam), anti-HIF-2α (ab199, Abcam), anti-β-actin (sc-47778, Santa Cruz Biotechnology).

Techniques: