anti phosphospecific stat3  (Cell Signaling Technology Inc)

Journal: Cancer Gene Therapy

Article Title: GATA3 suppresses human fibroblasts-induced metastasis of clear cell renal cell carcinoma via an anti-IL6/STAT3 mechanism

doi: 10.1038/s41417-019-0146-2

Figure Lengend Snippet: The cytokines secretion and STAT3 activation after the co-culture of NF-CM and ccRCC cells. CcRCC cells were cultured for 72 h in the presence or absence of different NF-CMs. a Expression levels of IL6, VEGF, IL1β, and CXCL12 in media were measured using ELISA, respectively. IL6 levels were remarkably increased in all cells, while VEGF was only increased in ACHN cells. CXCL12 secretion was increased in 786-O and 769-P cells, but IL1β secretion was decreased. * P < 0.05, ** P < 0.01, *** P < 0.005 vs. 1640. b Protein expression of p-STAT3 and total STAT3 were detected by western blotting, and representative results from one of the three independent experiments are presented. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. 1640. Note that NF-CM induced expression of IL6, VEGF and CXCL12 increased, decreased that of IL1β and activated STAT3 in ccRCC cells

Article Snippet: Anti-STAT3, anti-phosphospecific STAT3 (p-STAT3; Tyr705), anti-MMP2, anti-MMP9, anti-GAPDH, anti-E-cadherin, anti-α-SMA antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Co-Culture Assay, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer Gene Therapy

Article Title: GATA3 suppresses human fibroblasts-induced metastasis of clear cell renal cell carcinoma via an anti-IL6/STAT3 mechanism

doi: 10.1038/s41417-019-0146-2

Figure Lengend Snippet: The effects of IL6 on ccRCC cells migration and the IL6/STAT3 signaling pathway. CcRCC cells (769-P and ACHN) were treated with 1640 or 1640 + IL6 (200 μg/ml), NF-CM or NF-CM + anti-IL6 (50 μg/ml) for 48 h. a Cells migration ability was measured by transwell assays. Migration cells at 48-h time point were presented. *** P < 0.001 vs. 1640. Anti-IL6 antibody (50 μg/ml) inhibited the effect. ** P < 0.01, *** P < 0.001 vs. NF-CM. b P-STAT3, STAT3, MMP2, and MMP9 were analyzed using western blotting. Values represent the mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01 1640 vs. 1640 + IL6, NF-CM vs. NF-CM + anti-IL6. Note that recombinant human IL6 could promote ccRCC cells migration and activate STAT3 and MMP2, while anti-IL6 antibody could abolish the effect

Article Snippet: Anti-STAT3, anti-phosphospecific STAT3 (p-STAT3; Tyr705), anti-MMP2, anti-MMP9, anti-GAPDH, anti-E-cadherin, anti-α-SMA antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Migration, Western Blot, Recombinant

Journal: Cancer Gene Therapy

Article Title: GATA3 suppresses human fibroblasts-induced metastasis of clear cell renal cell carcinoma via an anti-IL6/STAT3 mechanism

doi: 10.1038/s41417-019-0146-2

Figure Lengend Snippet: GATA3-induced attenuation of p-STAT3 activation in ccRCC cells. The 769-P and ACHN cells were transfected with overexpression GATA3 plasmid or control vector for 48 h, and ( a ) the relative protein expressions of p-STAT3 and STAT3 were detected by western blotting. * P < 0.05 vs. vector. b 769-P/vector, 769-P/GATA3, and ACHN/vector ACHN/GATA3 were incubated with 200 μg/ml IL-6 for 48 h. The expression of p-STAT3 and STAT3 was detected by western blotting. * P < 0.05, ** P < 0.001 vs. vector. The representative results from one of the three independent experiments are presented. Note that GATA3 inhibited the IL6-induced STAT3 activation

Article Snippet: Anti-STAT3, anti-phosphospecific STAT3 (p-STAT3; Tyr705), anti-MMP2, anti-MMP9, anti-GAPDH, anti-E-cadherin, anti-α-SMA antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Control, Western Blot, Incubation, Expressing

Journal: Cancer Gene Therapy

Article Title: GATA3 suppresses human fibroblasts-induced metastasis of clear cell renal cell carcinoma via an anti-IL6/STAT3 mechanism

doi: 10.1038/s41417-019-0146-2

Figure Lengend Snippet: The schematic interaction of GATA3 and IL6 in the STAT3 activation and ccRCC cell metastasis

Article Snippet: Anti-STAT3, anti-phosphospecific STAT3 (p-STAT3; Tyr705), anti-MMP2, anti-MMP9, anti-GAPDH, anti-E-cadherin, anti-α-SMA antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Kaempferia parviflora Extract Inhibits STAT3 Activation and Interleukin-6 Production in HeLa Cervical Cancer Cells

doi: 10.3390/ijms20174226

Figure Lengend Snippet: ( A ) The effects of KP on suppressing EGF-induced expression of intracellular signaling proteins determined by Western blot analysis of cell lysates from HeLa cells exposed to KP extract at different concentrations; ( B ) quantitative analysis of cellular level of interleukin (IL)-6 protein; ( C ) quantitative analysis of cellular level of GP130 protein; ( D ) quantitative analysis of signal transducers and activators of transcription 3 (STAT3) phosphorylation; ( E ) quantitative analysis of cellular level of Mcl-1 protein. The protein expression levels of IL-6, GP130, and Mcl1 were normalized against actin, while the phosphorylated form of STAT3 was normalized against total STAT3 protein. Data represent mean ± SD of three independent experiments. * p < 0.05 in comparison with cells treated with DMSO + EGF.

Article Snippet: Primary antibodies included a 1:1000 dilution of a rabbit anti-IL-6 antibody (catalog number 12153), a rabbit anti-GP130 antibody (catalog number 3732), a mouse anti-STAT3 antibody (catalog number 9139), a rabbit anti-Mcl-1 antibody (catalog number 94296), a rabbit anti-EGFR antibody (catalog number 4267), a phosphospecific rabbit anti-STAT3 (Tyr705) antibody (catalog number 9145), phosphospecific rabbit anti-EGFR antibodies (Tyr1045 (catalog number 2237) and Tyr1068 (catalog number 3777)), a phosphospecific rabbit anti-ERK1/2 (Thr202/Tyr204) antibody (catalog number 4370), or a phosphospecific rabbit anti-Akt (Ser473) antibody (catalog number 4060), and a 1:10,000 dilution of a mouse anti-β-actin antibody (catalog number MA1115) (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Kaempferia parviflora Extract Inhibits STAT3 Activation and Interleukin-6 Production in HeLa Cervical Cancer Cells

doi: 10.3390/ijms20174226

Figure Lengend Snippet: The effects of KP on suppressing IL-6-induced STAT3 signaling in HeLa cells. ( A ) Immunofluorescence for nuclear STAT3 phosphorylation (red) in HeLa cells treated with KP at 15 μg/mL, with the presence of human recombinant IL-6 (50 ng/mL) for 24 h. The total STAT3 protein (green) was detected to visualize total expression of STAT3; ( B ) Western blotting and quantitative analysis for pSTAT3, Mcl-1, and pAkt in HeLa cells treated with different concentrations of KP or DMSO, with the presence of human recombinant IL-6 for 24 h. Micrographs were captured at 40× magnification. Data represent mean ± SD of three replicates. * p < 0.05.

Article Snippet: Primary antibodies included a 1:1000 dilution of a rabbit anti-IL-6 antibody (catalog number 12153), a rabbit anti-GP130 antibody (catalog number 3732), a mouse anti-STAT3 antibody (catalog number 9139), a rabbit anti-Mcl-1 antibody (catalog number 94296), a rabbit anti-EGFR antibody (catalog number 4267), a phosphospecific rabbit anti-STAT3 (Tyr705) antibody (catalog number 9145), phosphospecific rabbit anti-EGFR antibodies (Tyr1045 (catalog number 2237) and Tyr1068 (catalog number 3777)), a phosphospecific rabbit anti-ERK1/2 (Thr202/Tyr204) antibody (catalog number 4370), or a phosphospecific rabbit anti-Akt (Ser473) antibody (catalog number 4060), and a 1:10,000 dilution of a mouse anti-β-actin antibody (catalog number MA1115) (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Immunofluorescence, Phospho-proteomics, Recombinant, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation

doi: 10.3389/fimmu.2017.00926

Figure Lengend Snippet: Regulation of STAT3 pathway following lipopolysaccharide (LPS) treatment. (A) iDCs were treated with LPS 50 ng/ml or IL-6 150 ng/ml. STAT3 and STAT3-Tyr705 phosphorylation levels were assessed by Western blot up to 240 min. The blot presented is representative of two independent experiments. Quantification of the bands was plotted as phosphorylated STAT3/non-phosphorylated STAT3 ratio. (B) iDCs were pretreated with tocilizumab or ritux 50 µg/ml during 30 min and then treated with LPS 50 ng/ml or IL-6 150 ng/ml. STAT3 and STAT3-Tyr705 phosphorylation levels were assessed by Western blot at 120 min. The blot presented is representative of two independent experiments. (C,D) iDCs were pretreated with SB203580 (SB) or PD98059 (PD) 25 µM prior to LPS maturation (50 ng/ml). STAT3 and STAT3-Tyr705 phosphorylation levels (C) and Erk1/2-Thr202/Tyr204 and p38-Thr180/Tyr182 phosphorylation levels (D) were assessed by Western blot. The quantification was done on the correct respective actin blots as explained in Section “ .” The blot presented is representative of two independent experiments. Quantification of the bands was plotted as phosphorylated STAT3/total STAT3 (C) or phosphorylated Erk1/2 or p38/actin (D) ratio.

Article Snippet: Phosphospecific antibodies against STAT3 (Tyr705) (clone 3E2), p38 MAPK (Thr180/Tyr182), p42/44 MAPK (Erk1/2) (Thr202/Tyr204), and antibodies against the total form of STAT3 (clone 79D7), p38 MAPK, p42/44 MAPK, and β-actin were from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: Frontiers in Immunology

Article Title: Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation

doi: 10.3389/fimmu.2017.00926

Figure Lengend Snippet: Lipopolysaccharide (LPS)-induced mIL-6R loss desensitizes dendritic cells (DCs) to IL-6 treatment. (A) Flowchart: iDCs underwent LPS maturation 50 ng/ml for 48 h prior to stimulation by IL-6 150 ng/ml. Analyses were made at 15, 30, and 60 min. Control cells were maintained in immature state before to be stimulated as well with IL-6. (B) iDCs were treated as described in (A) . STAT3 and STAT3-Tyr705 phosphorylation levels were assessed by Western blot. The blot presented is representative of three independent experiments. Quantification of the bands was plotted as phosphorylated STAT3/non-phosphorylated STAT3 ratio.

Article Snippet: Phosphospecific antibodies against STAT3 (Tyr705) (clone 3E2), p38 MAPK (Thr180/Tyr182), p42/44 MAPK (Erk1/2) (Thr202/Tyr204), and antibodies against the total form of STAT3 (clone 79D7), p38 MAPK, p42/44 MAPK, and β-actin were from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Control, Phospho-proteomics, Western Blot

Journal: Molecular medicine reports

Article Title: SirT1 and STAT3 protect retinal pigmented epithelium cells against oxidative stress.

doi: 10.3892/mmr.2015.3570

Figure Lengend Snippet: Figure 1. Effects of oxidants H2O2 and oxLDL on RPEs. ARPE‑19 cells were grown at 80% confluence and exposed to H2O2 (50 µM) or oxLDL (200 µg/ml) for 24 h. Subsequently, (A) proliferation, (B) apoptosis, (C) intracellular ROS and (D) senescence of RPEs were assessed. The expression of SirT1 and STAT3 mRNA was evaluated by reverse transcription-quantitative polymerase chain reaction. Ct values were normalized using the 2‑∆∆Ct method as relative quantifica tion as shown in (E) SirT1 and (F) STAT3. Values are expressed as the mean ± standard deviation of three independent experiments. Analysis of variance was performed to analyze the differences statistically. *P<0.05, **P<0.01 vs. control. OxLDL, oxidized low density lipoprotein; RPEs, retinal pigmented epithelium cells; ROS, reactive oxygen species; mRNA, messenger RNA; SirT1, sirtuin 1; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The membranes were subsequently incubated with rabbit monoclonal primary antibodies against STAT3 (1:500; Minneapolis, MI, USA), phosphospecific STAT3 (Tyr705) (1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), SirT1 (1:500; Sigma-Aldrich) and mouse β-actin (1:1,000; Sigma‐Aldrich) overnight at 4 ̊C.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Control

Journal: Molecular medicine reports

Article Title: SirT1 and STAT3 protect retinal pigmented epithelium cells against oxidative stress.

doi: 10.3892/mmr.2015.3570

Figure Lengend Snippet: Figure 3. SirT1 counteracts the expression of STAT3 in RPEs during oxidative stress. ARPE‑19 cells were pretreated with 10 mM RSV (RSV group), small interfering RNA for SirT1KO group or C‑SirT1KO group. Subsequently, pretreated cells were exposed to H2O2 for 24 h. (A) Relative expression of STAT3 mRNA was detected by reverse transcription-quantitative polymerase chain reaction. (B) Western blot analysis demonstrated the efficiency of SirT1 knockdown, as well as the expression levels of STAT3 protein and pSTAT3 in the SirT1KO and C‑SirT1KO groups. (C) Silencing of STAT3 was confirmed by western blot analysis. (D) Expression levels of SirT1 were compared between the STAT3OV and C‑STAT3OV groups, and between the STAT3KO and C‑STAT3KO groups. Values are expressed as the mean ± standard deviation of three independent experiments, *P<0.05. SirT1, sirtuin 1; STAT3, signal transducer and activator of transcription 3; RSV, resveratrol; sirT1KO, SirT1 knockdown; C‑SirT1KO, non-target short hairpin RNA; STAT3OV, STAT3 overexpression; C‑STAT3OV, control expression vector; mRNA, messenger RNA; pSTAT3, phosphorylated STAT3.

Article Snippet: The membranes were subsequently incubated with rabbit monoclonal primary antibodies against STAT3 (1:500; Minneapolis, MI, USA), phosphospecific STAT3 (Tyr705) (1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), SirT1 (1:500; Sigma-Aldrich) and mouse β-actin (1:1,000; Sigma‐Aldrich) overnight at 4 ̊C.

Techniques: Expressing, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Standard Deviation, shRNA, Over Expression, Control, Plasmid Preparation

Journal: Molecular medicine reports

Article Title: SirT1 and STAT3 protect retinal pigmented epithelium cells against oxidative stress.

doi: 10.3892/mmr.2015.3570

Figure Lengend Snippet: Figure 4. STAT3 directly protects ARPE-19 cells from oxidative stress. ARPE‑19 cells were transfected with pRC/CMV‑STAT3 (STAT3OV group) or pcRC/CMV (C‑STAT3OV group). Cells were subsequently treated with H2O2 for 24 h following incubated with 5 mM SirT1 inhibitor NA for 4 h. (A) Proliferation, (B) apoptosis, (C) intracellular reactive oxygen species and (D) senescence of retinal pigmented epithelium cells were assessed and compared amongst these groups. Data in each panel are from three independent experiments. *P<0.05, **P<0.01. STAT3, signal transducer and activator of transcription 3; SirT1, sirtuin 1; NA, nicotinamide.

Article Snippet: The membranes were subsequently incubated with rabbit monoclonal primary antibodies against STAT3 (1:500; Minneapolis, MI, USA), phosphospecific STAT3 (Tyr705) (1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), SirT1 (1:500; Sigma-Aldrich) and mouse β-actin (1:1,000; Sigma‐Aldrich) overnight at 4 ̊C.

Techniques: Transfection, Incubation