anti stat3 phospho y705 antibody ep2147y  (Abcam)

 
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    Anti STAT3 phospho Y705 antibody EP2147Y
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    AB76315
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    Structured Review

    Abcam anti stat3 phospho y705 antibody ep2147y
    SMURF1, SHP-1, and <t>STAT3</t> expression and p-STAT3 levels in ectopic endometrial stromal cells from patients with endometriosis. (A,B) SMURF1, SHP-1, and p-STAT3 levels in ectopic endometrium from women with endometriosis (EM) or in normal endometrium from women without endometriosis (Nor) were measured by IHC. Scale bar: 25 µm. (C) Intracellular expression of cytoskeleton proteins CK19 and vimentin was measured with immunocytochemistry. Scale bar: 10 µm. (D) Stromal cells expressed vimentin and CK19 were measured by flow cytometric analysis. (E,F,G) Representative SMURF1, SHP-1, and STAT3 expression as well as p-STAT3 levels in endometrial stromal cells from ectopic endometrium of women with endometriosis (EMS1–3) or that from normal endometrium of women without endometriosis (Nor1–3) was measured by Western blotting. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using two-way ANOVA followed by Tukey’s test (B,F) or unpaired t -test (G). ***, P

    https://www.bioz.com/result/anti stat3 phospho y705 antibody ep2147y/product/Abcam
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    anti stat3 phospho y705 antibody ep2147y - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis"

    Article Title: SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis

    Journal: Annals of Translational Medicine

    doi: 10.21037/atm-20-2897

    SMURF1, SHP-1, and STAT3 expression and p-STAT3 levels in ectopic endometrial stromal cells from patients with endometriosis. (A,B) SMURF1, SHP-1, and p-STAT3 levels in ectopic endometrium from women with endometriosis (EM) or in normal endometrium from women without endometriosis (Nor) were measured by IHC. Scale bar: 25 µm. (C) Intracellular expression of cytoskeleton proteins CK19 and vimentin was measured with immunocytochemistry. Scale bar: 10 µm. (D) Stromal cells expressed vimentin and CK19 were measured by flow cytometric analysis. (E,F,G) Representative SMURF1, SHP-1, and STAT3 expression as well as p-STAT3 levels in endometrial stromal cells from ectopic endometrium of women with endometriosis (EMS1–3) or that from normal endometrium of women without endometriosis (Nor1–3) was measured by Western blotting. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using two-way ANOVA followed by Tukey’s test (B,F) or unpaired t -test (G). ***, P
    Figure Legend Snippet: SMURF1, SHP-1, and STAT3 expression and p-STAT3 levels in ectopic endometrial stromal cells from patients with endometriosis. (A,B) SMURF1, SHP-1, and p-STAT3 levels in ectopic endometrium from women with endometriosis (EM) or in normal endometrium from women without endometriosis (Nor) were measured by IHC. Scale bar: 25 µm. (C) Intracellular expression of cytoskeleton proteins CK19 and vimentin was measured with immunocytochemistry. Scale bar: 10 µm. (D) Stromal cells expressed vimentin and CK19 were measured by flow cytometric analysis. (E,F,G) Representative SMURF1, SHP-1, and STAT3 expression as well as p-STAT3 levels in endometrial stromal cells from ectopic endometrium of women with endometriosis (EMS1–3) or that from normal endometrium of women without endometriosis (Nor1–3) was measured by Western blotting. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using two-way ANOVA followed by Tukey’s test (B,F) or unpaired t -test (G). ***, P

    Techniques Used: Expressing, Immunohistochemistry, Immunocytochemistry, Western Blot

    Silencing of SMURF1 and/or SHP-1 regulates the proliferation and invasion of ectopic endometrial stromal cells. Ectopic endometrial stromal cells were infected with pLKO.1-SMURF1-shRNA, pLKO.1-SHP-1-shRNA, or pLKO.1-scramble shRNA as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. *, P
    Figure Legend Snippet: Silencing of SMURF1 and/or SHP-1 regulates the proliferation and invasion of ectopic endometrial stromal cells. Ectopic endometrial stromal cells were infected with pLKO.1-SMURF1-shRNA, pLKO.1-SHP-1-shRNA, or pLKO.1-scramble shRNA as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. *, P

    Techniques Used: Infection, shRNA, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

    Overexpression of SMURF1 and/or SHP-1 regulates the proliferation and invasion of normal endometrial stromal cells. Normal endometrial stromal cells were infected with pLVX-Puro-SMURF1, pLVX-Puro-SHP-1, or blank pLVX-Puro as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. ***, P
    Figure Legend Snippet: Overexpression of SMURF1 and/or SHP-1 regulates the proliferation and invasion of normal endometrial stromal cells. Normal endometrial stromal cells were infected with pLVX-Puro-SMURF1, pLVX-Puro-SHP-1, or blank pLVX-Puro as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. ***, P

    Techniques Used: Over Expression, Infection, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

    2) Product Images from "IL-10 secreted by cancer-associated macrophages regulates proliferation and invasion in gastric cancer cells via c-Met/STAT3 signaling"

    Article Title: IL-10 secreted by cancer-associated macrophages regulates proliferation and invasion in gastric cancer cells via c-Met/STAT3 signaling

    Journal: Oncology Reports

    doi: 10.3892/or.2019.7206

    Protein expression levels of c-Met/STAT3 signaling proteins in different treatment groups. Western blot analysis was used to confirm the effects of IL-10 on proteins associated with the c-Met/STAT3 pathway. *P
    Figure Legend Snippet: Protein expression levels of c-Met/STAT3 signaling proteins in different treatment groups. Western blot analysis was used to confirm the effects of IL-10 on proteins associated with the c-Met/STAT3 pathway. *P

    Techniques Used: Expressing, Western Blot

    3) Product Images from "Radiation induces an inflammatory response that results in STAT3-dependent changes in cellular plasticity and radioresistance of breast cancer stem-like cells"

    Article Title: Radiation induces an inflammatory response that results in STAT3-dependent changes in cellular plasticity and radioresistance of breast cancer stem-like cells

    Journal: International journal of radiation biology

    doi: 10.1080/09553002.2020.1705423

    STAT3 is required for ALDH activity and is activated upon treatment with radiation. (A) Sum159 cells were treated with siRNA to STAT3 or Control siRNA and ALDH activity was determined 48 hours following knockdown. (B C) Sum159 (B) and MDA-MB-231 (C) cells were treated with inhibitors to STAT3 and analyzed 48 hours after treatment for ALDH activity. (D E) Activation of JAK2 and STAT3 following 8 Gy radiation STAT3 was assessed via Western blot for increases in phosphorylation. (D). SUM159 cells or MD-MB-231 (E) were treated with 8 Gy of radiation for 24 hours prior to lysing. Blots were probed for phosphorylated STAT3, or phosphorylated JAK2. Beta-actin was used as a loading control. Relative protein levels of phosphorylated STAT3 were normalized to betaactin.
    Figure Legend Snippet: STAT3 is required for ALDH activity and is activated upon treatment with radiation. (A) Sum159 cells were treated with siRNA to STAT3 or Control siRNA and ALDH activity was determined 48 hours following knockdown. (B C) Sum159 (B) and MDA-MB-231 (C) cells were treated with inhibitors to STAT3 and analyzed 48 hours after treatment for ALDH activity. (D E) Activation of JAK2 and STAT3 following 8 Gy radiation STAT3 was assessed via Western blot for increases in phosphorylation. (D). SUM159 cells or MD-MB-231 (E) were treated with 8 Gy of radiation for 24 hours prior to lysing. Blots were probed for phosphorylated STAT3, or phosphorylated JAK2. Beta-actin was used as a loading control. Relative protein levels of phosphorylated STAT3 were normalized to betaactin.

    Techniques Used: Activity Assay, Multiple Displacement Amplification, Activation Assay, Western Blot

    Inhibition of STAT3 blocks radiation induced increases in ALDH activity in sorted cells. (A) Representative flow cytometry analysis of ALDH activity in ALDH-negative subpopulation following STAT3 inhibition and radiation treatment. SUM159 cells were sorted into ALDH negative subpopulation using FACS and cultured for two days to allow recovery from sorting. ALDH activity was measured by ALDEFLUOR assay and percentage of ALDH-positive cells was determined by flow cytometry. (B C) SUM159 cells were sorted into ALDH− (B) or ALDHþ (C) populations and were treated with 8 Gy of radiation and/or STAT3 inhibitors Stattic, or C188-9 and cultured for five days with inhibitors before reassessing ALDH activity. (D). Sum159 cells were treated with 8 Gy with and without Stattic. Cells were grown for 5 days in the presence of Stattic and RNA was obtained. Quantitative PCR was performed using primers to Nanog, Oct-4 and Sox-2. Graphs represents average of three separate experiments. Error bars represent standard deviation. * p
    Figure Legend Snippet: Inhibition of STAT3 blocks radiation induced increases in ALDH activity in sorted cells. (A) Representative flow cytometry analysis of ALDH activity in ALDH-negative subpopulation following STAT3 inhibition and radiation treatment. SUM159 cells were sorted into ALDH negative subpopulation using FACS and cultured for two days to allow recovery from sorting. ALDH activity was measured by ALDEFLUOR assay and percentage of ALDH-positive cells was determined by flow cytometry. (B C) SUM159 cells were sorted into ALDH− (B) or ALDHþ (C) populations and were treated with 8 Gy of radiation and/or STAT3 inhibitors Stattic, or C188-9 and cultured for five days with inhibitors before reassessing ALDH activity. (D). Sum159 cells were treated with 8 Gy with and without Stattic. Cells were grown for 5 days in the presence of Stattic and RNA was obtained. Quantitative PCR was performed using primers to Nanog, Oct-4 and Sox-2. Graphs represents average of three separate experiments. Error bars represent standard deviation. * p

    Techniques Used: Inhibition, Activity Assay, Flow Cytometry, FACS, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

    Inhibition of STAT3 sensitizes cells to radiation and decreases mammopshere forming ability. (A) Clonogenic analysis of SUM159 or MDA-MB-231 cells following treatment with 2,4 or 8 Gy of radiation, 1 lM Stattic, 5 lM C188-9, or a combination of radiation and inhibitor. Colonies ( > 50 cells) were counted and data is reported as colony-forming efficiency (colonies counted/cells plated). Error bars represent standard deviation. Inhibition of STAT3 reduced colony-forming ability and sensitized cells to radiation. *** p
    Figure Legend Snippet: Inhibition of STAT3 sensitizes cells to radiation and decreases mammopshere forming ability. (A) Clonogenic analysis of SUM159 or MDA-MB-231 cells following treatment with 2,4 or 8 Gy of radiation, 1 lM Stattic, 5 lM C188-9, or a combination of radiation and inhibitor. Colonies ( > 50 cells) were counted and data is reported as colony-forming efficiency (colonies counted/cells plated). Error bars represent standard deviation. Inhibition of STAT3 reduced colony-forming ability and sensitized cells to radiation. *** p

    Techniques Used: Inhibition, Multiple Displacement Amplification, Standard Deviation

    4) Product Images from "Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine"

    Article Title: Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine

    Journal: Biochimica et biophysica acta. General subjects

    doi: 10.1016/j.bbagen.2019.05.019

    ]. The measurements provide two indistinguishable PWM logos, suggesting an identical DNA-binding specificity for pY705 STAT3 and AcK685+pY705 STAT3.
    Figure Legend Snippet: ]. The measurements provide two indistinguishable PWM logos, suggesting an identical DNA-binding specificity for pY705 STAT3 and AcK685+pY705 STAT3.

    Techniques Used: Binding Assay

    . Average K D and Hill slope values are displayed ( n =3, ± SD). The affinity of non-phosphorylated STAT3 variants was below the detection limit. mP: millipolarization units.
    Figure Legend Snippet: . Average K D and Hill slope values are displayed ( n =3, ± SD). The affinity of non-phosphorylated STAT3 variants was below the detection limit. mP: millipolarization units.

    Techniques Used:

    Crystal structure of AcK685+pY705 STAT3 in a complex with DNA. A. Superposition of AcK685+pY705 STAT3 (cyan) and pY705 STAT3 (magenta, PDB ID: 1BG1). Top: overall structure. Bottom: residues 500–715, with residues AcK685, K685, and pY705 displayed in sticks model. B. Electron density map around residues AcK685 (top) and pY705 (bottom) (2 F o – F c at 1.0 σ and 0.7 σ level, respectively), displayed relative to the position of the same residues in non-acetylated pY705 STAT3 (magenta). C. Position and orientation of residues AcK685, K685, and pY705 (in sticks model) within the SH2 domain of Lys685-acetylated and non-acetylated STAT3.
    Figure Legend Snippet: Crystal structure of AcK685+pY705 STAT3 in a complex with DNA. A. Superposition of AcK685+pY705 STAT3 (cyan) and pY705 STAT3 (magenta, PDB ID: 1BG1). Top: overall structure. Bottom: residues 500–715, with residues AcK685, K685, and pY705 displayed in sticks model. B. Electron density map around residues AcK685 (top) and pY705 (bottom) (2 F o – F c at 1.0 σ and 0.7 σ level, respectively), displayed relative to the position of the same residues in non-acetylated pY705 STAT3 (magenta). C. Position and orientation of residues AcK685, K685, and pY705 (in sticks model) within the SH2 domain of Lys685-acetylated and non-acetylated STAT3.

    Techniques Used:

    5) Product Images from "CHI3L1 alleviate acute liver injury by inhibiting Th1 cells differentiation through STAT3 signaling pathway"

    Article Title: CHI3L1 alleviate acute liver injury by inhibiting Th1 cells differentiation through STAT3 signaling pathway

    Journal: Annals of Translational Medicine

    doi: 10.21037/atm-20-6127

    CHI3L1 promotes phosphorylation of the transcription factor STAT-3 via T-bet activation. (A) T-bet and GATA-3 expression in WT and CHI3L1-KO mice were determined by flow cytometry. (B) Expression of P-STAT3 in mRNA and protein levels were analyzed in different groups in ALI models. (C) CD4+CD62L+ T cells extracted from WT and CHI3L1-KO mice were induced to Th17, Th1, and Th2 cells for three days, and WP1033 was added to analyzed the role of STAT3 phosphorylation, the expression of IL-4, IL-17 and IFN-γ was detected by flow cytometry. The levels of serum ALT, AST (D), and mRNA expression of Foxp3 (E) in the liver from different groups are shown. Data are shown as means ± SEM from 3 independent experiments, and for in vivo experiments, each group includes 5 mice. NS, no significant; **, P
    Figure Legend Snippet: CHI3L1 promotes phosphorylation of the transcription factor STAT-3 via T-bet activation. (A) T-bet and GATA-3 expression in WT and CHI3L1-KO mice were determined by flow cytometry. (B) Expression of P-STAT3 in mRNA and protein levels were analyzed in different groups in ALI models. (C) CD4+CD62L+ T cells extracted from WT and CHI3L1-KO mice were induced to Th17, Th1, and Th2 cells for three days, and WP1033 was added to analyzed the role of STAT3 phosphorylation, the expression of IL-4, IL-17 and IFN-γ was detected by flow cytometry. The levels of serum ALT, AST (D), and mRNA expression of Foxp3 (E) in the liver from different groups are shown. Data are shown as means ± SEM from 3 independent experiments, and for in vivo experiments, each group includes 5 mice. NS, no significant; **, P

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Flow Cytometry, AST Assay, In Vivo

    6) Product Images from "Salidroside inhibits migration and invasion of poorly differentiated thyroid cancer cells"

    Article Title: Salidroside inhibits migration and invasion of poorly differentiated thyroid cancer cells

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.13096

    Salidroside inhibited the phosphorylation activation of JAK2 ‐STAT3 signaling pathway. ( a ) The phosphorylation of JAK2 and STAT3 were determined by western blot analysis and represented by the ratio of pJAK2/JAK2 and pSTAT3/STAT3. ( b ) The histogram below shows the results of the quantitative analysis of changes in phosphorylation of JAK2 and STAT3. Data are shown as the mean ± SD of three experiments. * P
    Figure Legend Snippet: Salidroside inhibited the phosphorylation activation of JAK2 ‐STAT3 signaling pathway. ( a ) The phosphorylation of JAK2 and STAT3 were determined by western blot analysis and represented by the ratio of pJAK2/JAK2 and pSTAT3/STAT3. ( b ) The histogram below shows the results of the quantitative analysis of changes in phosphorylation of JAK2 and STAT3. Data are shown as the mean ± SD of three experiments. * P

    Techniques Used: Activation Assay, Western Blot

    7) Product Images from "miR-221-3p and miR-222-3p regulate the SOCS3/STAT3 signaling pathway to downregulate the expression of NIS and reduce radiosensitivity in thyroid cancer"

    Article Title: miR-221-3p and miR-222-3p regulate the SOCS3/STAT3 signaling pathway to downregulate the expression of NIS and reduce radiosensitivity in thyroid cancer

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2021.10084

    miR-221-3p and miR-222-3p upregulate the protein expression levels of p-STAT3 and vimentin and downregulate the expression of NIS and E-cadherin. (A) BCPAP cells were transfected with miR-221-3p, miR-222-3p inhibitor and NC. FTC133 and TPC1 cells were infected with GFP-miR-221-3p, GFP-miR-222-3p or GFP lentiviral vectors. Western blotting was used to analyze the expression levels of p-STAT3, STAT3, vimentin, E-cadherin and NIS. Grouping of BCPAP images from different gels than those of FTC133 and TPC1. Grouping of FTC133 and TPC1 images from different parts of the same gel. Bands of p-STAT3 and STAT3 from different gels. (B-D) Relative expression levels of p-STAT3/STAT3, vimentin/GAPDH, E-cadherin/GAPDH and NIS/GAPDH were analyzed using ImageJ 7.0 software in each cell model. Each band was measured in triplicate. ** P
    Figure Legend Snippet: miR-221-3p and miR-222-3p upregulate the protein expression levels of p-STAT3 and vimentin and downregulate the expression of NIS and E-cadherin. (A) BCPAP cells were transfected with miR-221-3p, miR-222-3p inhibitor and NC. FTC133 and TPC1 cells were infected with GFP-miR-221-3p, GFP-miR-222-3p or GFP lentiviral vectors. Western blotting was used to analyze the expression levels of p-STAT3, STAT3, vimentin, E-cadherin and NIS. Grouping of BCPAP images from different gels than those of FTC133 and TPC1. Grouping of FTC133 and TPC1 images from different parts of the same gel. Bands of p-STAT3 and STAT3 from different gels. (B-D) Relative expression levels of p-STAT3/STAT3, vimentin/GAPDH, E-cadherin/GAPDH and NIS/GAPDH were analyzed using ImageJ 7.0 software in each cell model. Each band was measured in triplicate. ** P

    Techniques Used: Expressing, Transfection, Infection, Western Blot, Software

    8) Product Images from "Low Levels of Sox2 are required for Melanoma Tumor-Repopulating Cell Dormancy"

    Article Title: Low Levels of Sox2 are required for Melanoma Tumor-Repopulating Cell Dormancy

    Journal: Theranostics

    doi: 10.7150/thno.29698

    Dormancy exit is mediated via STAT3 and p53 activation. (A) p53 , p27 , or p21 expression was quantified by real-time PCR. Total mRNAs of control B16 cells, B16 cells transfected with Sox2 shRNA, Sox2-KO cells, or cells treated with IFN-γ (100 ng/ml) or treated with Etoposide (a DNA re-ligation inhibitor, 50 μM) were extracted. The values represent mean ± SEM from three independent experiments. (B) Western blotting analysis of p53, p27, or p21 protein level in control B16 cells, B16 cells transfected with Sox2 shRNA, or Sox2 KO cells from three independent experiments. (C) Sox2 knockout cells, with or without the p53 inhibitor, Pifithrin (2 μM), were treated with 0.1% DMSO or Tazarotene (100 μM) for 3 days in 90-Pa fibrin gels. The cell apoptotic ratio was measured by flow cytometry. The values represent mean ± SEM from three independent experiments. (D) Representative images and quantitative analysis of S-STAT3 (phospho-S727), Y-STAT3 (phospho-Y705), or total STAT3 expression in control B16 cells, B16 cells transfected with Sox2 shRNA, or Sox2-KO cells by western blotting. The values represent mean ± SEM from three independent experiments. N.S. : not statistically significant. (E, F) ChIP (chromatin immunoprecipitation) assays were performed using normal rabbit IgG (negative control), S-STAT3, or Y-STAT3 antibody on control Sox2 shRNA and Sox2-KO cell lysates. Two sets of primers were used for p27 and p53 promoter regions. Relative enrichment was determined by real time PCR. The values represent mean ± SEM ( n = 3). * P
    Figure Legend Snippet: Dormancy exit is mediated via STAT3 and p53 activation. (A) p53 , p27 , or p21 expression was quantified by real-time PCR. Total mRNAs of control B16 cells, B16 cells transfected with Sox2 shRNA, Sox2-KO cells, or cells treated with IFN-γ (100 ng/ml) or treated with Etoposide (a DNA re-ligation inhibitor, 50 μM) were extracted. The values represent mean ± SEM from three independent experiments. (B) Western blotting analysis of p53, p27, or p21 protein level in control B16 cells, B16 cells transfected with Sox2 shRNA, or Sox2 KO cells from three independent experiments. (C) Sox2 knockout cells, with or without the p53 inhibitor, Pifithrin (2 μM), were treated with 0.1% DMSO or Tazarotene (100 μM) for 3 days in 90-Pa fibrin gels. The cell apoptotic ratio was measured by flow cytometry. The values represent mean ± SEM from three independent experiments. (D) Representative images and quantitative analysis of S-STAT3 (phospho-S727), Y-STAT3 (phospho-Y705), or total STAT3 expression in control B16 cells, B16 cells transfected with Sox2 shRNA, or Sox2-KO cells by western blotting. The values represent mean ± SEM from three independent experiments. N.S. : not statistically significant. (E, F) ChIP (chromatin immunoprecipitation) assays were performed using normal rabbit IgG (negative control), S-STAT3, or Y-STAT3 antibody on control Sox2 shRNA and Sox2-KO cell lysates. Two sets of primers were used for p27 and p53 promoter regions. Relative enrichment was determined by real time PCR. The values represent mean ± SEM ( n = 3). * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, shRNA, Ligation, Western Blot, Knock-Out, Flow Cytometry, Chromatin Immunoprecipitation, Negative Control

    9) Product Images from "Talin1 knockdown prohibits the proliferation and migration of colorectal cancer cells via the EMT signaling pathway"

    Article Title: Talin1 knockdown prohibits the proliferation and migration of colorectal cancer cells via the EMT signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10902

    Talin1 knockdown affects the expression of proteins associated with the EMT signaling pathway in HCT116 cells. (A) E-cadherin and vimentin protein levels were measured by western blotting in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control. (B) E-cadherin protein levels in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control (n=3). (C) Vimentin protein levels in in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control (n=3). (D) E-cadherin mRNA levels in tumor and normal tissues. (E) ZO-1 mRNA levels in tumor and normal tissues. (F) Occludin mRNA levels in tumor and normal tissues. (G) pSTAT3 and total STAT3 protein levels measured by western blotting. (H) pSTAT3 and total STAT3 protein levels in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control (n=3). (I) IL-6 mRNA levels in blood samples from patients and healthy controls. (J) Serum IL-6 concentrations in blood samples from patients and healthy controls. (K) IL-6 mRNA levels in tumor and adjacent normal tissues. Data are presented as mean ± SEM. *P
    Figure Legend Snippet: Talin1 knockdown affects the expression of proteins associated with the EMT signaling pathway in HCT116 cells. (A) E-cadherin and vimentin protein levels were measured by western blotting in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control. (B) E-cadherin protein levels in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control (n=3). (C) Vimentin protein levels in in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control (n=3). (D) E-cadherin mRNA levels in tumor and normal tissues. (E) ZO-1 mRNA levels in tumor and normal tissues. (F) Occludin mRNA levels in tumor and normal tissues. (G) pSTAT3 and total STAT3 protein levels measured by western blotting. (H) pSTAT3 and total STAT3 protein levels in talin1-knockdown HCT116 cell lines and HCT116 cells transfected with the shRNA control (n=3). (I) IL-6 mRNA levels in blood samples from patients and healthy controls. (J) Serum IL-6 concentrations in blood samples from patients and healthy controls. (K) IL-6 mRNA levels in tumor and adjacent normal tissues. Data are presented as mean ± SEM. *P

    Techniques Used: Expressing, Western Blot, Transfection, shRNA

    10) Product Images from "Inhibition of miR-155-5p attenuates the valvular damage induced by rheumatic heart disease"

    Article Title: Inhibition of miR-155-5p attenuates the valvular damage induced by rheumatic heart disease

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4420

    Reverse transcription-quantitative PCR and western blot analysis. (A) The relative mRNA expression of S1PR1, SOCS1 and STAT3 in the four groups. (B) Western blot analysis of S1PR1, SOCS1, STAT3 and p-STAT3 in the four groups. (C) The relative protein expression of S1PR1, SOCS1, STAT3 and p-STAT3 in the four groups. (D) The ratio of phosphorylated vs. total protein for STAT3. This figure shows that expression of S1PR1 and SOCS1 was increased and expression of p-STAT3 was decreased after AAV-injection. Data are shown as the mean ± standard deviation; * P
    Figure Legend Snippet: Reverse transcription-quantitative PCR and western blot analysis. (A) The relative mRNA expression of S1PR1, SOCS1 and STAT3 in the four groups. (B) Western blot analysis of S1PR1, SOCS1, STAT3 and p-STAT3 in the four groups. (C) The relative protein expression of S1PR1, SOCS1, STAT3 and p-STAT3 in the four groups. (D) The ratio of phosphorylated vs. total protein for STAT3. This figure shows that expression of S1PR1 and SOCS1 was increased and expression of p-STAT3 was decreased after AAV-injection. Data are shown as the mean ± standard deviation; * P

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Injection, Standard Deviation

    Immunohistochemistry analysis of S1PR1, STAT3 and p-STAT3 in valve tissues. (A) Immunohistochemistry for S1PR1, STAT3 and p-STAT3 in valve tissues; magnification, ×400; the arrows represent positive cells. (B) The IHS. (C) The positive cells percentage. This figure shows that expression of S1PR1 was increased and expression of p-STAT3 was decreased after AAV-injection. (D) The ratio of phosphorylated vs. total protein for STAT3 (ISH score). (E) The ratio of phosphorylated vs. total protein for STAT3 (positive cells percentage). Data are shown as the mean ± standard deviation; * P
    Figure Legend Snippet: Immunohistochemistry analysis of S1PR1, STAT3 and p-STAT3 in valve tissues. (A) Immunohistochemistry for S1PR1, STAT3 and p-STAT3 in valve tissues; magnification, ×400; the arrows represent positive cells. (B) The IHS. (C) The positive cells percentage. This figure shows that expression of S1PR1 was increased and expression of p-STAT3 was decreased after AAV-injection. (D) The ratio of phosphorylated vs. total protein for STAT3 (ISH score). (E) The ratio of phosphorylated vs. total protein for STAT3 (positive cells percentage). Data are shown as the mean ± standard deviation; * P

    Techniques Used: Immunohistochemistry, Expressing, Injection, In Situ Hybridization, Standard Deviation

    11) Product Images from "HOTAIR/miR-17-5p Axis is Involved in the Propofol-Mediated Cardioprotection Against Ischemia/Reperfusion Injury"

    Article Title: HOTAIR/miR-17-5p Axis is Involved in the Propofol-Mediated Cardioprotection Against Ischemia/Reperfusion Injury

    Journal: Clinical Interventions in Aging

    doi: 10.2147/CIA.S286429

    The effects of HOTAIR, STAT3, and PPF on H/R-induced apoptosis of H9c2 cells. ( A–G ) Apoptosis of H9c2 cells was detected by flow cytometry after HOTAIR or STAT3 was selectively regulated (N=3). ** P
    Figure Legend Snippet: The effects of HOTAIR, STAT3, and PPF on H/R-induced apoptosis of H9c2 cells. ( A–G ) Apoptosis of H9c2 cells was detected by flow cytometry after HOTAIR or STAT3 was selectively regulated (N=3). ** P

    Techniques Used: Flow Cytometry

    The effect of PPF treatment on rats with MIRI and its effect on HOTAIR and miR-17-5p expression. ( A ) CK-MB in the serum of the rats in each group was detected with ELISA (N=3). ( B ) LDH in the serum of the rats in each group was detected with ELISA (N=3). ( C ) qRT-PCR was used to detect the expression level of HOTAIR in the heart tissue of the rats (N=3). ( D ) MiR-17-5p expression in heart tissue of the rats was detected by qRT-PCR. ( E and F ) Western blot was used to detect by the expression of p-STAT3 and STAT3 in heart tissue of the rats. ** P
    Figure Legend Snippet: The effect of PPF treatment on rats with MIRI and its effect on HOTAIR and miR-17-5p expression. ( A ) CK-MB in the serum of the rats in each group was detected with ELISA (N=3). ( B ) LDH in the serum of the rats in each group was detected with ELISA (N=3). ( C ) qRT-PCR was used to detect the expression level of HOTAIR in the heart tissue of the rats (N=3). ( D ) MiR-17-5p expression in heart tissue of the rats was detected by qRT-PCR. ( E and F ) Western blot was used to detect by the expression of p-STAT3 and STAT3 in heart tissue of the rats. ** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

    H/R treatment regulated miR-17-5p and STAT3 expressions via HOTAIR. ( A ) Empty plasmids and HOTAIR overexpression plasmids were transfected into H9c2 cells treated with H/R, respectively, and the expression of miR-17-5p was detected by qRT-PCR (N=3). ( B and C ) The expressions of STAT3 and p-STAT3 in H9c2 cells were detected by Western blot (N=3). ( D ) Control siRNA and HOTAIR siRNA were transfected into H9c2 cells treated with H/R and PPF, and the expression of miR-17-5p was detected by qRT-PCR (N=3). ( E and F ) The expressions of STAT3 and p-STAT3 in H9c2 cells were detected by Western blot (N=3). ** P
    Figure Legend Snippet: H/R treatment regulated miR-17-5p and STAT3 expressions via HOTAIR. ( A ) Empty plasmids and HOTAIR overexpression plasmids were transfected into H9c2 cells treated with H/R, respectively, and the expression of miR-17-5p was detected by qRT-PCR (N=3). ( B and C ) The expressions of STAT3 and p-STAT3 in H9c2 cells were detected by Western blot (N=3). ( D ) Control siRNA and HOTAIR siRNA were transfected into H9c2 cells treated with H/R and PPF, and the expression of miR-17-5p was detected by qRT-PCR (N=3). ( E and F ) The expressions of STAT3 and p-STAT3 in H9c2 cells were detected by Western blot (N=3). ** P

    Techniques Used: Over Expression, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    The effects of PPF on HOTAIR, miR-17-5p, and STAT3 expressions in H9c2 cells. ( A and B ) The expression levels of HOTAIR and miR-17-5p in H9c2 cells were detected by qRT-PCR (N=3). ( C and D ) Western blot was used to detect the expressions of STAT3 and p-STAT3 in H9c2 cells (N=3). * P
    Figure Legend Snippet: The effects of PPF on HOTAIR, miR-17-5p, and STAT3 expressions in H9c2 cells. ( A and B ) The expression levels of HOTAIR and miR-17-5p in H9c2 cells were detected by qRT-PCR (N=3). ( C and D ) Western blot was used to detect the expressions of STAT3 and p-STAT3 in H9c2 cells (N=3). * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    The effects of HOTAIR on the protective function of PPF in MIRI rats. ( A ) CK-MB in the serum of the rats in each group was detected with ELISA (N=3). ( B ) LDH in the serum of the rats in each group was detected with ELISA (N=3). ( C ) The expression level of miR-17-5p in the myocardial tissue of rats was detected by qRT-PCR (N=3). ( D and E ) Western blot was used to detect the expressions of STAT3 and p-STAT3 protein in cardiac tissue of the rats (N=3). * P
    Figure Legend Snippet: The effects of HOTAIR on the protective function of PPF in MIRI rats. ( A ) CK-MB in the serum of the rats in each group was detected with ELISA (N=3). ( B ) LDH in the serum of the rats in each group was detected with ELISA (N=3). ( C ) The expression level of miR-17-5p in the myocardial tissue of rats was detected by qRT-PCR (N=3). ( D and E ) Western blot was used to detect the expressions of STAT3 and p-STAT3 protein in cardiac tissue of the rats (N=3). * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot

    12) Product Images from "Gallic acid suppresses colon cancer proliferation by inhibiting SRC and EGFR phosphorylation"

    Article Title: Gallic acid suppresses colon cancer proliferation by inhibiting SRC and EGFR phosphorylation

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2021.10070

    Effects of GA on p-SRC and p-STAT3 level. The effects of GA in (A) HCT116 and (B) HT29 colon cancer cells were analyzed by immunofluorescence. n=3. Magnification, x400. ** P
    Figure Legend Snippet: Effects of GA on p-SRC and p-STAT3 level. The effects of GA in (A) HCT116 and (B) HT29 colon cancer cells were analyzed by immunofluorescence. n=3. Magnification, x400. ** P

    Techniques Used: Immunofluorescence

    GA decreases SRC and EGFR phosphorylation level to inhibit STAT3 and AKT phosphorylation. (A) The proteins expression in HCT116 tumor tissues. (B) The proteins expression in HT29 tumor tissues. The level of p-SRC/SRC, p-EGFR/EGFR, p-STAT3/STAT3 and p-AKT/AKT in tumor tissues were analyzed by western blotting. β-actin was used as the internal reference gene. n=6. * P
    Figure Legend Snippet: GA decreases SRC and EGFR phosphorylation level to inhibit STAT3 and AKT phosphorylation. (A) The proteins expression in HCT116 tumor tissues. (B) The proteins expression in HT29 tumor tissues. The level of p-SRC/SRC, p-EGFR/EGFR, p-STAT3/STAT3 and p-AKT/AKT in tumor tissues were analyzed by western blotting. β-actin was used as the internal reference gene. n=6. * P

    Techniques Used: Expressing, Western Blot

    13) Product Images from "Long non-coding RNA MAFG-AS1 knockdown blocks malignant progression in breast cancer cells by inactivating JAK2/STAT3 signaling pathway via MAFG-AS1/miR-3196/TFAP2A axis"

    Article Title: Long non-coding RNA MAFG-AS1 knockdown blocks malignant progression in breast cancer cells by inactivating JAK2/STAT3 signaling pathway via MAFG-AS1/miR-3196/TFAP2A axis

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    JAK2/STAT3 signaling pathway is involved in the MAFG-AS1/miR-3196/TFAP2A regulatory axis. T47D and MDA-MB-231 cells were transfected with si-MAFG-AS1#3, si-MAFG-AS1#3+anti-miR-3196 or si-MAFG-AS1#3+pcDNA-TFAP2A, si-NC, si-MAFG-AS1#3+anti-miR-NC or si-MAFG-AS1#3+pcDNA acting as the control, respectively. A and B. The relative protein levels of TFAP2A, p-JAK2/JAK2 and p-STAT3/STAT3 were measured by western blot. * P
    Figure Legend Snippet: JAK2/STAT3 signaling pathway is involved in the MAFG-AS1/miR-3196/TFAP2A regulatory axis. T47D and MDA-MB-231 cells were transfected with si-MAFG-AS1#3, si-MAFG-AS1#3+anti-miR-3196 or si-MAFG-AS1#3+pcDNA-TFAP2A, si-NC, si-MAFG-AS1#3+anti-miR-NC or si-MAFG-AS1#3+pcDNA acting as the control, respectively. A and B. The relative protein levels of TFAP2A, p-JAK2/JAK2 and p-STAT3/STAT3 were measured by western blot. * P

    Techniques Used: Multiple Displacement Amplification, Transfection, Western Blot

    14) Product Images from "SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis"

    Article Title: SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis

    Journal: Annals of Translational Medicine

    doi: 10.21037/atm-20-2897

    SMURF1, SHP-1, and STAT3 expression and p-STAT3 levels in ectopic endometrial stromal cells from patients with endometriosis. (A,B) SMURF1, SHP-1, and p-STAT3 levels in ectopic endometrium from women with endometriosis (EM) or in normal endometrium from women without endometriosis (Nor) were measured by IHC. Scale bar: 25 µm. (C) Intracellular expression of cytoskeleton proteins CK19 and vimentin was measured with immunocytochemistry. Scale bar: 10 µm. (D) Stromal cells expressed vimentin and CK19 were measured by flow cytometric analysis. (E,F,G) Representative SMURF1, SHP-1, and STAT3 expression as well as p-STAT3 levels in endometrial stromal cells from ectopic endometrium of women with endometriosis (EMS1–3) or that from normal endometrium of women without endometriosis (Nor1–3) was measured by Western blotting. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using two-way ANOVA followed by Tukey’s test (B,F) or unpaired t -test (G). ***, P
    Figure Legend Snippet: SMURF1, SHP-1, and STAT3 expression and p-STAT3 levels in ectopic endometrial stromal cells from patients with endometriosis. (A,B) SMURF1, SHP-1, and p-STAT3 levels in ectopic endometrium from women with endometriosis (EM) or in normal endometrium from women without endometriosis (Nor) were measured by IHC. Scale bar: 25 µm. (C) Intracellular expression of cytoskeleton proteins CK19 and vimentin was measured with immunocytochemistry. Scale bar: 10 µm. (D) Stromal cells expressed vimentin and CK19 were measured by flow cytometric analysis. (E,F,G) Representative SMURF1, SHP-1, and STAT3 expression as well as p-STAT3 levels in endometrial stromal cells from ectopic endometrium of women with endometriosis (EMS1–3) or that from normal endometrium of women without endometriosis (Nor1–3) was measured by Western blotting. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using two-way ANOVA followed by Tukey’s test (B,F) or unpaired t -test (G). ***, P

    Techniques Used: Expressing, Immunohistochemistry, Immunocytochemistry, Western Blot

    Silencing of SMURF1 and/or SHP-1 regulates the proliferation and invasion of ectopic endometrial stromal cells. Ectopic endometrial stromal cells were infected with pLKO.1-SMURF1-shRNA, pLKO.1-SHP-1-shRNA, or pLKO.1-scramble shRNA as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. *, P
    Figure Legend Snippet: Silencing of SMURF1 and/or SHP-1 regulates the proliferation and invasion of ectopic endometrial stromal cells. Ectopic endometrial stromal cells were infected with pLKO.1-SMURF1-shRNA, pLKO.1-SHP-1-shRNA, or pLKO.1-scramble shRNA as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. *, P

    Techniques Used: Infection, shRNA, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

    Overexpression of SMURF1 and/or SHP-1 regulates the proliferation and invasion of normal endometrial stromal cells. Normal endometrial stromal cells were infected with pLVX-Puro-SMURF1, pLVX-Puro-SHP-1, or blank pLVX-Puro as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. ***, P
    Figure Legend Snippet: Overexpression of SMURF1 and/or SHP-1 regulates the proliferation and invasion of normal endometrial stromal cells. Normal endometrial stromal cells were infected with pLVX-Puro-SMURF1, pLVX-Puro-SHP-1, or blank pLVX-Puro as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. ***, P

    Techniques Used: Over Expression, Infection, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

    15) Product Images from "COP9 Signalosome Subunit 3 Restricts Neuroinflammatory Responses During Cerebral Ischemia/Reperfusion Injury Through Stabilizing Suppressor of Cytokine Signaling 3 Protein"

    Article Title: COP9 Signalosome Subunit 3 Restricts Neuroinflammatory Responses During Cerebral Ischemia/Reperfusion Injury Through Stabilizing Suppressor of Cytokine Signaling 3 Protein

    Journal: Neuropsychiatric Disease and Treatment

    doi: 10.2147/NDT.S298966

    SOCS3 suppresses OGD/R-induced STAT3 activation and inflammatory factor expression in BV-2 microglial cells. ( A ) SOCS3 protein expression was examined by Western blot in OGD/R-treated BV2 microglia. GAPDH protein expression was used as loading control. ( B ) Cellular distribution of SOCS3 protein (Red) were detected by immunofluorescence in OGD/R-treated BV2 microglia. Nuclei were stained with DAPI (blue). ( C ) SOCS3 mRNA expression was examined by qPCR in BV2 microglia transfected with the indicated siRNAs or plasmids. ( D ) IL6 mRNA expression was examined by qPCR in BV2 microglia transfected with the indicated siRNAs or plasmids under OGD/R condition. ( E ) Protein expressions of STAT3 and phosphorylated STAT3 (p-STAT3) were examined by Western blot in BV2 microglia transfected with pCMV3-N-FLAG-SOCS3 plasmid under OGD/R condition. ( F ) mRNA expressions of Cyclin D1 and MCL1 were examined by qPCR in BV2 microglia transfected with pCMV3-N-FLAG-SOCS3 plasmid under OGD/R condition. Data are represented as means ± SEM (n=3; *Represents P
    Figure Legend Snippet: SOCS3 suppresses OGD/R-induced STAT3 activation and inflammatory factor expression in BV-2 microglial cells. ( A ) SOCS3 protein expression was examined by Western blot in OGD/R-treated BV2 microglia. GAPDH protein expression was used as loading control. ( B ) Cellular distribution of SOCS3 protein (Red) were detected by immunofluorescence in OGD/R-treated BV2 microglia. Nuclei were stained with DAPI (blue). ( C ) SOCS3 mRNA expression was examined by qPCR in BV2 microglia transfected with the indicated siRNAs or plasmids. ( D ) IL6 mRNA expression was examined by qPCR in BV2 microglia transfected with the indicated siRNAs or plasmids under OGD/R condition. ( E ) Protein expressions of STAT3 and phosphorylated STAT3 (p-STAT3) were examined by Western blot in BV2 microglia transfected with pCMV3-N-FLAG-SOCS3 plasmid under OGD/R condition. ( F ) mRNA expressions of Cyclin D1 and MCL1 were examined by qPCR in BV2 microglia transfected with pCMV3-N-FLAG-SOCS3 plasmid under OGD/R condition. Data are represented as means ± SEM (n=3; *Represents P

    Techniques Used: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

    CSN3 restricting OGD/R-induced STAT3 activation and inflammatory responses dependents on SOCS3. ( A ) IL6 mRNA expression was examined by qPCR in BV2 microglia transfected with pCMV3-N-Myc-CSN3. ( B ) IL6 mRNA expression was examined by qPCR in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and then treated with ODG/R. ( C ) Protein expressions of STAT3 and p-STAT3 were examined by Western blot in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and then treated with ODG/R. ( D ) Protein expressions of STAT3 and p-STAT3 were examined by Western blot in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and si-SOCS3, and then treated with ODG/R. ( E ) mRNA expressions of Cyclin D1 and MCL1 were examined by qPCR in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and si-SOCS3, and then treated with ODG/R. Data are represented as means ± SEM (n=3; *Represents P
    Figure Legend Snippet: CSN3 restricting OGD/R-induced STAT3 activation and inflammatory responses dependents on SOCS3. ( A ) IL6 mRNA expression was examined by qPCR in BV2 microglia transfected with pCMV3-N-Myc-CSN3. ( B ) IL6 mRNA expression was examined by qPCR in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and then treated with ODG/R. ( C ) Protein expressions of STAT3 and p-STAT3 were examined by Western blot in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and then treated with ODG/R. ( D ) Protein expressions of STAT3 and p-STAT3 were examined by Western blot in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and si-SOCS3, and then treated with ODG/R. ( E ) mRNA expressions of Cyclin D1 and MCL1 were examined by qPCR in BV2 microglia transfected with pCMV3-N-Myc-CSN3 and si-SOCS3, and then treated with ODG/R. Data are represented as means ± SEM (n=3; *Represents P

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

    16) Product Images from "Da Cheng Qi Decoction Alleviates Cerulein-Stimulated AR42J Pancreatic Acinar Cell Injury via the JAK2/STAT3 Signaling Pathway"

    Article Title: Da Cheng Qi Decoction Alleviates Cerulein-Stimulated AR42J Pancreatic Acinar Cell Injury via the JAK2/STAT3 Signaling Pathway

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2021/6657036

    DCQD suppressed JAK2/STAT3 signaling pathway in a concentration-dependent manner. (a) Real-time PCR was performed to detect the mRNA expression of JAK2 and STAT3. (b) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. (c) Immunofluorescence staining (400×) was performed to detect the location of p-STAT3 in cells. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3.Immunofluorescence micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P
    Figure Legend Snippet: DCQD suppressed JAK2/STAT3 signaling pathway in a concentration-dependent manner. (a) Real-time PCR was performed to detect the mRNA expression of JAK2 and STAT3. (b) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. (c) Immunofluorescence staining (400×) was performed to detect the location of p-STAT3 in cells. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3.Immunofluorescence micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining

    Effect of DCQD on downstream effectors in the JAK2/STAT3 pathway. (a) Real-time PCR was performed to detect the mRNA expression of Bax and Bcl-XL. (b) Western blot was performed to detect the protein level of p-STAT3, Bax, and Bcl-XL. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. All bar graphics: # P
    Figure Legend Snippet: Effect of DCQD on downstream effectors in the JAK2/STAT3 pathway. (a) Real-time PCR was performed to detect the mRNA expression of Bax and Bcl-XL. (b) Western blot was performed to detect the protein level of p-STAT3, Bax, and Bcl-XL. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. All bar graphics: # P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot

    The amelioration of DCQD was mediated by suppressing the activation of JAK2/STAT3 signaling pathway. (a and f) The morphological changes of cells were assessed by using an optical microscope (400×). The releases of amylase, lipase, and C-reactive protein were detected by amylase assay kit (b and g), lipase assay kit (c and h), and C-reactive protein assay kit (d and i), respectively. (e) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. GAPDH was as the loading control for band density normalization. (j) Western blot was performed to detect the protein level of STAT3 and p-STAT3. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. Micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P
    Figure Legend Snippet: The amelioration of DCQD was mediated by suppressing the activation of JAK2/STAT3 signaling pathway. (a and f) The morphological changes of cells were assessed by using an optical microscope (400×). The releases of amylase, lipase, and C-reactive protein were detected by amylase assay kit (b and g), lipase assay kit (c and h), and C-reactive protein assay kit (d and i), respectively. (e) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. GAPDH was as the loading control for band density normalization. (j) Western blot was performed to detect the protein level of STAT3 and p-STAT3. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. Micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P

    Techniques Used: Activation Assay, Microscopy, Western Blot

    17) Product Images from "Defective dystrophic thymus determines degenerative changes in skeletal muscle"

    Article Title: Defective dystrophic thymus determines degenerative changes in skeletal muscle

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22305-x

    Cellularity, NF-kB/STATs expression, and autophagy in thymus of C57Bl and mdx mice. FACS analysis of thymus homogenate from mdx and C57Bl mice at 8 weeks of age demonstrates no significant alteration of T cells ( a , b ), and few differences in CD4−CD8−DN stages, in particular DN3 (CD44−CD25+) and DN4 (CD44+CD25+) ( c ). The number of TCRβ+CD69+ cells ( d ) and of Foxp3+CD25+ cells ( e ) was significantly increased in thymus of mdx mice. Cropped image of a representative WB and densitometric analysis revealed a downregulation of NF-kB, IKKi, and STAT3 in mdx thymus ( f ). RT-qPCR of p62 expression is shown in g . Autophagy markers such as Atg7, p62 and LC3 were also assessed by WB analysis. Representative WB image and quantification of LC3-II/LC3-I showed the impairment of the autophagic flux ( h ). All protein expression was normalized on actin, as a loading control. The comparisons between the averages of the groups were evaluated using two-sided Student’s t -test. c * p = 0.0177 (DN3), * p = 0.0351 (DN4). d * p = 0.043. e * p = 0.0332. f ** p = 0.0048 (NF-κB), ** p = 0.0018 (IKKi). h ** p = 0.0026. Data are presented as mean ± SD of three independent experiments with n = 7 (mdx) and n = 5 (C57Bl) mice ( a – e ); n = 6 mice/group ( f , h ); n = 3 mice (mdx) and n = 6 mice (C57Bl) ( g ). Source data are provided as a Source Data file.
    Figure Legend Snippet: Cellularity, NF-kB/STATs expression, and autophagy in thymus of C57Bl and mdx mice. FACS analysis of thymus homogenate from mdx and C57Bl mice at 8 weeks of age demonstrates no significant alteration of T cells ( a , b ), and few differences in CD4−CD8−DN stages, in particular DN3 (CD44−CD25+) and DN4 (CD44+CD25+) ( c ). The number of TCRβ+CD69+ cells ( d ) and of Foxp3+CD25+ cells ( e ) was significantly increased in thymus of mdx mice. Cropped image of a representative WB and densitometric analysis revealed a downregulation of NF-kB, IKKi, and STAT3 in mdx thymus ( f ). RT-qPCR of p62 expression is shown in g . Autophagy markers such as Atg7, p62 and LC3 were also assessed by WB analysis. Representative WB image and quantification of LC3-II/LC3-I showed the impairment of the autophagic flux ( h ). All protein expression was normalized on actin, as a loading control. The comparisons between the averages of the groups were evaluated using two-sided Student’s t -test. c * p = 0.0177 (DN3), * p = 0.0351 (DN4). d * p = 0.043. e * p = 0.0332. f ** p = 0.0048 (NF-κB), ** p = 0.0018 (IKKi). h ** p = 0.0026. Data are presented as mean ± SD of three independent experiments with n = 7 (mdx) and n = 5 (C57Bl) mice ( a – e ); n = 6 mice/group ( f , h ); n = 3 mice (mdx) and n = 6 mice (C57Bl) ( g ). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Mouse Assay, FACS, Western Blot, Quantitative RT-PCR

    18) Product Images from "SENP1-mediated deSUMOylation of JAK2 regulates its kinase activity and platinum drug resistance"

    Article Title: SENP1-mediated deSUMOylation of JAK2 regulates its kinase activity and platinum drug resistance

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-021-03635-6

    JAK2 SUMOylation is required for STAT3 phosphorylation. A Schematic diagram of the distribution of putative JAK2 SUMOylation sites, predicted by SUMOplot™. The score indicates the probability. B JAK2-SUMO mutant clones 1 and 2 fails to be SUMOylated in vitro. Recombinant His-JAK2 proteins were used for in vitro SUMOylation assay as described in “Materials and Methods”. C JAK2-SUMO mutant phosphorylates STAT3 (Tyr705) in vitro. Empty vector, His-JAK2 and His-JAK2 SUMO mutant were purified from HEK293T cells, followed by in vitro kinase assay as in Fig. 2F . D JAK2-SUMO mutant accumulates in cytoplasm. Empty vector, His-JAK2 and His-JAK2 SUMO mutant were transfected in IGROV1 cells, followed by immunofluorescence assay using anti-His antibody. Scale bar: 5 µm. Right panel, expression of JAK2 and JAK2-SUMO mutant. E Quantification of cellular distribution of JAK2 in IGROV1 cells as shown in ( D ). In total, 100 cells were counted in each sample. ***, p
    Figure Legend Snippet: JAK2 SUMOylation is required for STAT3 phosphorylation. A Schematic diagram of the distribution of putative JAK2 SUMOylation sites, predicted by SUMOplot™. The score indicates the probability. B JAK2-SUMO mutant clones 1 and 2 fails to be SUMOylated in vitro. Recombinant His-JAK2 proteins were used for in vitro SUMOylation assay as described in “Materials and Methods”. C JAK2-SUMO mutant phosphorylates STAT3 (Tyr705) in vitro. Empty vector, His-JAK2 and His-JAK2 SUMO mutant were purified from HEK293T cells, followed by in vitro kinase assay as in Fig. 2F . D JAK2-SUMO mutant accumulates in cytoplasm. Empty vector, His-JAK2 and His-JAK2 SUMO mutant were transfected in IGROV1 cells, followed by immunofluorescence assay using anti-His antibody. Scale bar: 5 µm. Right panel, expression of JAK2 and JAK2-SUMO mutant. E Quantification of cellular distribution of JAK2 in IGROV1 cells as shown in ( D ). In total, 100 cells were counted in each sample. ***, p

    Techniques Used: Mutagenesis, Clone Assay, In Vitro, Recombinant, Plasmid Preparation, Purification, Kinase Assay, Transfection, Immunofluorescence, Expressing

    19) Product Images from "Neuritin inhibits astrogliosis to ameliorate diabetic cognitive dysfunction"

    Article Title: Neuritin inhibits astrogliosis to ameliorate diabetic cognitive dysfunction

    Journal: Journal of Molecular Endocrinology

    doi: 10.1530/JME-20-0321

    Neuritin inhibits JAK2/STAT3 signaling pathway in U-118MG cells. Data are given as mean ± s.d. ( n = 3). * P
    Figure Legend Snippet: Neuritin inhibits JAK2/STAT3 signaling pathway in U-118MG cells. Data are given as mean ± s.d. ( n = 3). * P

    Techniques Used:

    Neuritin suppressed JAK2/STAT3 signaling pathway in the hippocampus. Phosphorylation level of JAK2 was measured using immunofluorescence in hippocampus (A) and its quantification of fluorescence integrated intensity (B). Phosphorylation level of STAT3 was measured using immunofluorescence (C) and its quantification of fluorescence integrated intensity (D). Mean ± s.d. , n = 6. * P
    Figure Legend Snippet: Neuritin suppressed JAK2/STAT3 signaling pathway in the hippocampus. Phosphorylation level of JAK2 was measured using immunofluorescence in hippocampus (A) and its quantification of fluorescence integrated intensity (B). Phosphorylation level of STAT3 was measured using immunofluorescence (C) and its quantification of fluorescence integrated intensity (D). Mean ± s.d. , n = 6. * P

    Techniques Used: Immunofluorescence, Fluorescence

    20) Product Images from "High density of CD68+ tumor-associated macrophages predicts a poor prognosis in gastric cancer mediated by IL-6 expression"

    Article Title: High density of CD68+ tumor-associated macrophages predicts a poor prognosis in gastric cancer mediated by IL-6 expression

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8119

    Levels of STAT3, P-STAT3 and IRF4 proteins were increased in the monocytes stimulated by M-CSF and IL-6. Levels of STAT3, P-STAT3 and IRF4 protein in the M-CSF group and M-CSF+IL-6 group were detected by western blotting. β-actin was used as the loading control. The assays were repeated in duplicate. STAT3, signal transducer and activator of transcription 3; pSTAT3, phosphorylated STAT3; M-CSF, macrophage colony stimulation factor; IL, interleukin, IRF4, interferon-regulatory factor 4.
    Figure Legend Snippet: Levels of STAT3, P-STAT3 and IRF4 proteins were increased in the monocytes stimulated by M-CSF and IL-6. Levels of STAT3, P-STAT3 and IRF4 protein in the M-CSF group and M-CSF+IL-6 group were detected by western blotting. β-actin was used as the loading control. The assays were repeated in duplicate. STAT3, signal transducer and activator of transcription 3; pSTAT3, phosphorylated STAT3; M-CSF, macrophage colony stimulation factor; IL, interleukin, IRF4, interferon-regulatory factor 4.

    Techniques Used: Western Blot

    21) Product Images from "Astragaloside IV Protects 6-Hydroxydopamine-Induced SH-SY5Y Cell Model of Parkinson’s Disease via Activating the JAK2/STAT3 Pathway"

    Article Title: Astragaloside IV Protects 6-Hydroxydopamine-Induced SH-SY5Y Cell Model of Parkinson’s Disease via Activating the JAK2/STAT3 Pathway

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2021.631501

    AS-IV activated the JAK2/STAT3 pathway in 6-OHDA-treated SH-SY5Y cells. Levels of JAK2, p-JAK2, STAT3, and p-STAT3 were detected using Western blot analysis. Each experiment was repeated for three times independently. Data are presented as mean ± standard deviation. One-way ANOVA was employed for the comparisons among multiple groups and Tukey’s multiple comparison test was applied for the post hoc test, ∗ p
    Figure Legend Snippet: AS-IV activated the JAK2/STAT3 pathway in 6-OHDA-treated SH-SY5Y cells. Levels of JAK2, p-JAK2, STAT3, and p-STAT3 were detected using Western blot analysis. Each experiment was repeated for three times independently. Data are presented as mean ± standard deviation. One-way ANOVA was employed for the comparisons among multiple groups and Tukey’s multiple comparison test was applied for the post hoc test, ∗ p

    Techniques Used: Western Blot, Standard Deviation

    AS-IV (100 μM) protected 6-OHDA-treated SH-SY5Y cells via activating the JAK2/STAT3 pathway. The JAK2/STAT3 pathway inhibitor SC99 (15 mM) was added to the cells treated with AS-IV (100 μM), with PBS as the control. (A) Levels of p-JAK2/JAK2 and p-STAT3/STAT3 were detected using Western blot analysis. (B) Viability of 6-OHDA-treated SH-SY5Y cells was detected using MTT assay. (C) Apoptosis rate of 6-OHDA-treated SH-SY5Y cells was detected flow cytometry. (C) Levels of IL-1β, IL-6, and TNF-α in 6-OHDA-treated SH-SY5Y cells were detected using the ELISA kits. (D) Changes of MDA, ROS and SOD levels in 6-OHDA-treated SH-SY5Y cells were detected using corresponding kits. Each experiment was repeated for three times independently. Data are presented as mean ± standard deviation. Data in panels (A–F) were analyzed using t test, ** p
    Figure Legend Snippet: AS-IV (100 μM) protected 6-OHDA-treated SH-SY5Y cells via activating the JAK2/STAT3 pathway. The JAK2/STAT3 pathway inhibitor SC99 (15 mM) was added to the cells treated with AS-IV (100 μM), with PBS as the control. (A) Levels of p-JAK2/JAK2 and p-STAT3/STAT3 were detected using Western blot analysis. (B) Viability of 6-OHDA-treated SH-SY5Y cells was detected using MTT assay. (C) Apoptosis rate of 6-OHDA-treated SH-SY5Y cells was detected flow cytometry. (C) Levels of IL-1β, IL-6, and TNF-α in 6-OHDA-treated SH-SY5Y cells were detected using the ELISA kits. (D) Changes of MDA, ROS and SOD levels in 6-OHDA-treated SH-SY5Y cells were detected using corresponding kits. Each experiment was repeated for three times independently. Data are presented as mean ± standard deviation. Data in panels (A–F) were analyzed using t test, ** p

    Techniques Used: Western Blot, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Standard Deviation

    22) Product Images from "Nicotine Upregulates the Level of Mcl-1 through STAT3 in H1299 Cells"

    Article Title: Nicotine Upregulates the Level of Mcl-1 through STAT3 in H1299 Cells

    Journal: Journal of Cancer

    doi: 10.7150/jca.35453

    Nicotine can induce the activation of STAT3. (A) H1299 cells were treated with 10µM nicotine for 0h, 0.5h, 1h, 2h and 4h. Western blot was done to check p-Y705 STAT3, p-S727 STAT3 and STAT3. (B) 10µM nicotine treated H1299 cells for 2h and the the immunofluorescence was done to check the subcellular localization of STAT3. Untreated cells were done as control.
    Figure Legend Snippet: Nicotine can induce the activation of STAT3. (A) H1299 cells were treated with 10µM nicotine for 0h, 0.5h, 1h, 2h and 4h. Western blot was done to check p-Y705 STAT3, p-S727 STAT3 and STAT3. (B) 10µM nicotine treated H1299 cells for 2h and the the immunofluorescence was done to check the subcellular localization of STAT3. Untreated cells were done as control.

    Techniques Used: Activation Assay, Western Blot, Immunofluorescence

    Activated STAT3 induced by nicotine regulates Mcl-1. (A) H1299 cells were transiently transfected with siRNA targeting STAT3 (STAT3-siRNA) and negative control siRNA (NC-siRNA) at least 24h, and then treated with 10µM nicotine for 0h, 1h and 2h. Western blot was done to check the expression of Mcl-1 and STAT3. (B) H1299 cells were treated in the absence or presence of increasing concentration of JSI-124 for 30min, and then treated with 10µM nicotine for 2h. Western blot analysis was performed to detect the p-Y705 STAT3 and the expression of Mcl-1. (C) The sequence of Mcl-1 promoter containing a STAT3 binding site. (D) H1299 cells were transiently transfected with EV, WT, mutant luciferase reporter plasmid and Rellina luciferase plasmid, then treated in the absence or presence of increasing concentration of nicotine for 2h. Samples were measured by a dual-luciferase reporter assay system. Each experiment was performed at least in triplicate. EV: original pGL3-Basic plasmid; WT: a 325bp long human Mcl-1 promoter fragment was inserted into pGL3-Basic plasmid; mutant: the Mcl-1 promoter fragment without the STAT3 binding site was inserted into pGL3-Basic plasmid.
    Figure Legend Snippet: Activated STAT3 induced by nicotine regulates Mcl-1. (A) H1299 cells were transiently transfected with siRNA targeting STAT3 (STAT3-siRNA) and negative control siRNA (NC-siRNA) at least 24h, and then treated with 10µM nicotine for 0h, 1h and 2h. Western blot was done to check the expression of Mcl-1 and STAT3. (B) H1299 cells were treated in the absence or presence of increasing concentration of JSI-124 for 30min, and then treated with 10µM nicotine for 2h. Western blot analysis was performed to detect the p-Y705 STAT3 and the expression of Mcl-1. (C) The sequence of Mcl-1 promoter containing a STAT3 binding site. (D) H1299 cells were transiently transfected with EV, WT, mutant luciferase reporter plasmid and Rellina luciferase plasmid, then treated in the absence or presence of increasing concentration of nicotine for 2h. Samples were measured by a dual-luciferase reporter assay system. Each experiment was performed at least in triplicate. EV: original pGL3-Basic plasmid; WT: a 325bp long human Mcl-1 promoter fragment was inserted into pGL3-Basic plasmid; mutant: the Mcl-1 promoter fragment without the STAT3 binding site was inserted into pGL3-Basic plasmid.

    Techniques Used: Transfection, Negative Control, Western Blot, Expressing, Concentration Assay, Sequencing, Binding Assay, Mutagenesis, Luciferase, Plasmid Preparation, Reporter Assay

    Nicotine may stimulate STAT3 in a JAKs-independent manner and the phosphorylation at Y705 site isn't indispensable for nuclear accumulation of STAT3. (A) H1299 cells were treated with 10µM nicotine in the absence or presence of increasing concentrations of AG490 for 2h. Phosphosylation of STAT3 and expression of Mcl-1 were determined by Western blot. (B) Immunofluorescence was done to check the subcellular localization of STAT3 using STAT3-antibody. H1299 cells were transiently transfected with Y705W-STAT3 plasmid (with Flag tag) at least 24h and then immunofluorescence was done with Flag-antibody. Y705W STAT3: the residue Y at 705 site of STAT3 was mutated to W.
    Figure Legend Snippet: Nicotine may stimulate STAT3 in a JAKs-independent manner and the phosphorylation at Y705 site isn't indispensable for nuclear accumulation of STAT3. (A) H1299 cells were treated with 10µM nicotine in the absence or presence of increasing concentrations of AG490 for 2h. Phosphosylation of STAT3 and expression of Mcl-1 were determined by Western blot. (B) Immunofluorescence was done to check the subcellular localization of STAT3 using STAT3-antibody. H1299 cells were transiently transfected with Y705W-STAT3 plasmid (with Flag tag) at least 24h and then immunofluorescence was done with Flag-antibody. Y705W STAT3: the residue Y at 705 site of STAT3 was mutated to W.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, FLAG-tag

    23) Product Images from "Hyperforin Ameliorates Imiquimod-Induced Psoriasis-Like Murine Skin Inflammation by Modulating IL-17A–Producing γδ T Cells"

    Article Title: Hyperforin Ameliorates Imiquimod-Induced Psoriasis-Like Murine Skin Inflammation by Modulating IL-17A–Producing γδ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.635076

    Hyperforin inhibits phosphorylation of MAPK and STAT3 pathway components in in vitro cultured γδ T cells. (A) Representative images of Western blot. (B–I) quantification of the Western blot data by densitometric analysis and normalization to GAPDH (n = 3 independent experiments). **P
    Figure Legend Snippet: Hyperforin inhibits phosphorylation of MAPK and STAT3 pathway components in in vitro cultured γδ T cells. (A) Representative images of Western blot. (B–I) quantification of the Western blot data by densitometric analysis and normalization to GAPDH (n = 3 independent experiments). **P

    Techniques Used: In Vitro, Cell Culture, Western Blot

    Hyperforin inhibits phosphorylation of MAPK and STAT3 pathway components induced by TNF-α in HaCaT cells. HaCaT cells were pretreated with different doses of hyperforin, and stimulated with TNF-α. The total protein was extracted from the cells and associated protein expression was determined via western blotting (A) . The quantification data are shown in the right panel (B–I) . (n = 3 independent experiments). **P
    Figure Legend Snippet: Hyperforin inhibits phosphorylation of MAPK and STAT3 pathway components induced by TNF-α in HaCaT cells. HaCaT cells were pretreated with different doses of hyperforin, and stimulated with TNF-α. The total protein was extracted from the cells and associated protein expression was determined via western blotting (A) . The quantification data are shown in the right panel (B–I) . (n = 3 independent experiments). **P

    Techniques Used: Expressing, Western Blot

    24) Product Images from "Vitamin D protects glomerular mesangial cells from high glucose-induced injury by repressing JAK/STAT signaling"

    Article Title: Vitamin D protects glomerular mesangial cells from high glucose-induced injury by repressing JAK/STAT signaling

    Journal: International Urology and Nephrology

    doi: 10.1007/s11255-020-02728-z

    VD inhibited tyrosine phosphorylation of JAK2, STAT1 and STAT3 which was induced by HG. Rat glomerular mesangial cells were cultured under different conditions with or without siVDR transfection for 24 or 48 h, and then subjected to immunoblotting
    Figure Legend Snippet: VD inhibited tyrosine phosphorylation of JAK2, STAT1 and STAT3 which was induced by HG. Rat glomerular mesangial cells were cultured under different conditions with or without siVDR transfection for 24 or 48 h, and then subjected to immunoblotting

    Techniques Used: Cell Culture, Transfection

    25) Product Images from "Fraxetin Inhibits the Proliferation and Metastasis of Glioma Cells by Inactivating JAK2/STAT3 Signaling"

    Article Title: Fraxetin Inhibits the Proliferation and Metastasis of Glioma Cells by Inactivating JAK2/STAT3 Signaling

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2021/5540139

    Fraxetin inhibits JAK2/STAT3 signaling in U251 cells. ((a), (b), and (c)) Western blot analysis of key proteins involved in JAK2/STAT3 signaling. Cells were treated with fraxetin (0 μ M, 100 μ M, and 200 μ M) for 24 h. (d) Immunofluorescence analysis of STAT3. Cells were treated with fraxetin (0 μ M, 100 μ M, and 200 μ M) for 24 h. ((e) and (f)) qRT-PCR analysis of IL-6 and IL-10. Cells were treated with fraxetin (0 μ M, 100 μ M, and 200 μ M) for 24 h. (g) CCK8 assay. Cells were treated with fraxetin (200 μ M) and Colivelin (10 μ M) together or fraxetin (200 μ M) alone for 24 h. (h) Transwell invasive assay. Cells were treated with fraxetin (200 μ M) and Colivelin (10 μ M) together or fraxetin (200 μ M) alone for 24 h. ∗ P
    Figure Legend Snippet: Fraxetin inhibits JAK2/STAT3 signaling in U251 cells. ((a), (b), and (c)) Western blot analysis of key proteins involved in JAK2/STAT3 signaling. Cells were treated with fraxetin (0 μ M, 100 μ M, and 200 μ M) for 24 h. (d) Immunofluorescence analysis of STAT3. Cells were treated with fraxetin (0 μ M, 100 μ M, and 200 μ M) for 24 h. ((e) and (f)) qRT-PCR analysis of IL-6 and IL-10. Cells were treated with fraxetin (0 μ M, 100 μ M, and 200 μ M) for 24 h. (g) CCK8 assay. Cells were treated with fraxetin (200 μ M) and Colivelin (10 μ M) together or fraxetin (200 μ M) alone for 24 h. (h) Transwell invasive assay. Cells were treated with fraxetin (200 μ M) and Colivelin (10 μ M) together or fraxetin (200 μ M) alone for 24 h. ∗ P

    Techniques Used: Western Blot, Immunofluorescence, Quantitative RT-PCR, CCK-8 Assay

    Fraxetin inhibits the growth of glioma xenografts in nude mice. (a) Picture of nude mice in control group and fraxetin-treatment group. (b) Tumor volume in control group and fraxetin-treatment group. (c) Picture of tumor in control group and fraxetin-treatment group. (d) Tumor weight of nude mice in control group and fraxetin-treatment group. ((e), (f), and (g)) Western blot analysis of key proteins involved in JAK2/STAT3 signaling in tumor. ((h) and (i)) histological analyses of Ki67 and cleaved caspase 3. ∗∗ P
    Figure Legend Snippet: Fraxetin inhibits the growth of glioma xenografts in nude mice. (a) Picture of nude mice in control group and fraxetin-treatment group. (b) Tumor volume in control group and fraxetin-treatment group. (c) Picture of tumor in control group and fraxetin-treatment group. (d) Tumor weight of nude mice in control group and fraxetin-treatment group. ((e), (f), and (g)) Western blot analysis of key proteins involved in JAK2/STAT3 signaling in tumor. ((h) and (i)) histological analyses of Ki67 and cleaved caspase 3. ∗∗ P

    Techniques Used: Mouse Assay, Western Blot

    26) Product Images from "Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies"

    Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

    Journal: The Analyst

    doi: 10.1039/d0an00030b

    Nuclear pSTAT3/STAT3 and β-hex release among RBL-2H3 clones. (A) pSTAT3/STAT3 measured per cell for the initial fragments’ biopsied after 7 days in culture (5 days on quad microraft array and 2 days’ post-biopsy in microwell collection array): i.) for the 6 individual target clones where each data point represents a single cell’s pSTAT3/STAT3 value and the mean value is plotted as a horizontal line (7–53 cells per fragment). ii.) the total distribution of pSTAT3/STAT3 per cell in the colony fragments pooled as low (L1, L2, and L3) and high (H1, H2, and H3). (B) Distribution of pSTAT3/STAT3 measured per cell for the resampled and expanded mother colonies after 18 days in culture (18 days’ post-cell seeding on microraft arrays): i.) for the 6 individual resampled clones (2,369 – 7,072 cells per clone) and ii.) for the resampled clones pooled based on their initial sort criteria of low (L1, L2, and L3) and high (H1, H2, and H3) pSTAT3/STAT3. (C) Percent β-hex release measured after activation via IgE cross-linking of FcεRI: i) in unsorted bulk RBL-2H3 cells without treatment and with activation of FcεRI cross-linking (N=12 wells per condition, 100,000 cells per well), and ii) in the expanded mother clones with activation of FcεRI cross-linking after 24–28 days in culture (N=12 wells per colony type, 100,000 cells per well). For each violin plot, the solid horizontal line represents the median value, while dotted horizontal lines below and above the medium value represent the 25 th and 75 th percentile values, respectively. p values are represented as p
    Figure Legend Snippet: Nuclear pSTAT3/STAT3 and β-hex release among RBL-2H3 clones. (A) pSTAT3/STAT3 measured per cell for the initial fragments’ biopsied after 7 days in culture (5 days on quad microraft array and 2 days’ post-biopsy in microwell collection array): i.) for the 6 individual target clones where each data point represents a single cell’s pSTAT3/STAT3 value and the mean value is plotted as a horizontal line (7–53 cells per fragment). ii.) the total distribution of pSTAT3/STAT3 per cell in the colony fragments pooled as low (L1, L2, and L3) and high (H1, H2, and H3). (B) Distribution of pSTAT3/STAT3 measured per cell for the resampled and expanded mother colonies after 18 days in culture (18 days’ post-cell seeding on microraft arrays): i.) for the 6 individual resampled clones (2,369 – 7,072 cells per clone) and ii.) for the resampled clones pooled based on their initial sort criteria of low (L1, L2, and L3) and high (H1, H2, and H3) pSTAT3/STAT3. (C) Percent β-hex release measured after activation via IgE cross-linking of FcεRI: i) in unsorted bulk RBL-2H3 cells without treatment and with activation of FcεRI cross-linking (N=12 wells per condition, 100,000 cells per well), and ii) in the expanded mother clones with activation of FcεRI cross-linking after 24–28 days in culture (N=12 wells per colony type, 100,000 cells per well). For each violin plot, the solid horizontal line represents the median value, while dotted horizontal lines below and above the medium value represent the 25 th and 75 th percentile values, respectively. p values are represented as p

    Techniques Used: Clone Assay, Activation Assay

    27) Product Images from "Da Cheng Qi Decoction Alleviates Cerulein-Stimulated AR42J Pancreatic Acinar Cell Injury via the JAK2/STAT3 Signaling Pathway"

    Article Title: Da Cheng Qi Decoction Alleviates Cerulein-Stimulated AR42J Pancreatic Acinar Cell Injury via the JAK2/STAT3 Signaling Pathway

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2021/6657036

    DCQD suppressed JAK2/STAT3 signaling pathway in a concentration-dependent manner. (a) Real-time PCR was performed to detect the mRNA expression of JAK2 and STAT3. (b) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. (c) Immunofluorescence staining (400×) was performed to detect the location of p-STAT3 in cells. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3.Immunofluorescence micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P
    Figure Legend Snippet: DCQD suppressed JAK2/STAT3 signaling pathway in a concentration-dependent manner. (a) Real-time PCR was performed to detect the mRNA expression of JAK2 and STAT3. (b) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. (c) Immunofluorescence staining (400×) was performed to detect the location of p-STAT3 in cells. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3.Immunofluorescence micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining

    Effect of DCQD on downstream effectors in the JAK2/STAT3 pathway. (a) Real-time PCR was performed to detect the mRNA expression of Bax and Bcl-XL. (b) Western blot was performed to detect the protein level of p-STAT3, Bax, and Bcl-XL. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. All bar graphics: # P
    Figure Legend Snippet: Effect of DCQD on downstream effectors in the JAK2/STAT3 pathway. (a) Real-time PCR was performed to detect the mRNA expression of Bax and Bcl-XL. (b) Western blot was performed to detect the protein level of p-STAT3, Bax, and Bcl-XL. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. All bar graphics: # P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot

    The amelioration of DCQD was mediated by suppressing the activation of JAK2/STAT3 signaling pathway. (a and f) The morphological changes of cells were assessed by using an optical microscope (400×). The releases of amylase, lipase, and C-reactive protein were detected by amylase assay kit (b and g), lipase assay kit (c and h), and C-reactive protein assay kit (d and i), respectively. (e) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. GAPDH was as the loading control for band density normalization. (j) Western blot was performed to detect the protein level of STAT3 and p-STAT3. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. Micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P
    Figure Legend Snippet: The amelioration of DCQD was mediated by suppressing the activation of JAK2/STAT3 signaling pathway. (a and f) The morphological changes of cells were assessed by using an optical microscope (400×). The releases of amylase, lipase, and C-reactive protein were detected by amylase assay kit (b and g), lipase assay kit (c and h), and C-reactive protein assay kit (d and i), respectively. (e) Western blot was performed to detect the protein level of JAK2, p-JAK2, STAT3, and p-STAT3. GAPDH was as the loading control for band density normalization. (j) Western blot was performed to detect the protein level of STAT3 and p-STAT3. GAPDH was as the loading control for band density normalization. Data are presented as the means ± SD, n = 3. Micrographs from one representative experiment out of three independent experiments are shown. All bar graphics: # P

    Techniques Used: Activation Assay, Microscopy, Western Blot

    28) Product Images from "Neuritin inhibits astrogliosis to ameliorate diabetic cognitive dysfunction"

    Article Title: Neuritin inhibits astrogliosis to ameliorate diabetic cognitive dysfunction

    Journal: Journal of Molecular Endocrinology

    doi: 10.1530/JME-20-0321

    Neuritin inhibits JAK2/STAT3 signaling pathway in U-118MG cells. Data are given as mean ± s.d. ( n = 3). * P
    Figure Legend Snippet: Neuritin inhibits JAK2/STAT3 signaling pathway in U-118MG cells. Data are given as mean ± s.d. ( n = 3). * P

    Techniques Used:

    Neuritin suppressed JAK2/STAT3 signaling pathway in the hippocampus. Phosphorylation level of JAK2 was measured using immunofluorescence in hippocampus (A) and its quantification of fluorescence integrated intensity (B). Phosphorylation level of STAT3 was measured using immunofluorescence (C) and its quantification of fluorescence integrated intensity (D). Mean ± s.d. , n = 6. * P
    Figure Legend Snippet: Neuritin suppressed JAK2/STAT3 signaling pathway in the hippocampus. Phosphorylation level of JAK2 was measured using immunofluorescence in hippocampus (A) and its quantification of fluorescence integrated intensity (B). Phosphorylation level of STAT3 was measured using immunofluorescence (C) and its quantification of fluorescence integrated intensity (D). Mean ± s.d. , n = 6. * P

    Techniques Used: Immunofluorescence, Fluorescence

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    Immunohistochemistry:

    Article Title: Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma
    Article Snippet: Mice were treated with LY5 for 30 days, and bioluminescent imaging (after IP luciferin administration) was performed to determine pulmonary metastatic disease load. .. To investigate the pharmacodynamics (PD) effects of LY5, pSTAT3 (p-Tyr705, ab76315, Abcam) and total STAT3 (ab119352, Abcam) were evaluated in pulmonary metastatic lesions using IHC in lung samples from mice euthanized 24 hrs after their last dose. .. Ex vivo pulmonary metastasis assay (PuMA) Murine K7M2 and human MG63.3 OS cells were treated with increasing concentrations of LY5 for 2 hrs or 7 hrs.

    Mouse Assay:

    Article Title: Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma
    Article Snippet: Mice were treated with LY5 for 30 days, and bioluminescent imaging (after IP luciferin administration) was performed to determine pulmonary metastatic disease load. .. To investigate the pharmacodynamics (PD) effects of LY5, pSTAT3 (p-Tyr705, ab76315, Abcam) and total STAT3 (ab119352, Abcam) were evaluated in pulmonary metastatic lesions using IHC in lung samples from mice euthanized 24 hrs after their last dose. .. Ex vivo pulmonary metastasis assay (PuMA) Murine K7M2 and human MG63.3 OS cells were treated with increasing concentrations of LY5 for 2 hrs or 7 hrs.

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    Abcam pstat3
    LY5 inhibits STAT3 phosphorylation in lung metastases but does not inhibit lung metastasis formation. (A-B) IHC staining of lung metastasis from mice treated with LY5. The metastatic tumor sections were stained with <t>pSTAT3</t> or hematoxylin and eosin. Western blotting was performed to confirm inhibition of STAT3 phosphorylation in lung tumor sections. (C) Mice were administered OS-17 cells stably expressing luciferase by tail vein injection and subsequently treated with LY5. After 30 days, imaging was performed to evaluate the effects of LY5 on the development of pulmonary metastasis.
    Pstat3, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LY5 inhibits STAT3 phosphorylation in lung metastases but does not inhibit lung metastasis formation. (A-B) IHC staining of lung metastasis from mice treated with LY5. The metastatic tumor sections were stained with pSTAT3 or hematoxylin and eosin. Western blotting was performed to confirm inhibition of STAT3 phosphorylation in lung tumor sections. (C) Mice were administered OS-17 cells stably expressing luciferase by tail vein injection and subsequently treated with LY5. After 30 days, imaging was performed to evaluate the effects of LY5 on the development of pulmonary metastasis.

    Journal: PLoS ONE

    Article Title: Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma

    doi: 10.1371/journal.pone.0181885

    Figure Lengend Snippet: LY5 inhibits STAT3 phosphorylation in lung metastases but does not inhibit lung metastasis formation. (A-B) IHC staining of lung metastasis from mice treated with LY5. The metastatic tumor sections were stained with pSTAT3 or hematoxylin and eosin. Western blotting was performed to confirm inhibition of STAT3 phosphorylation in lung tumor sections. (C) Mice were administered OS-17 cells stably expressing luciferase by tail vein injection and subsequently treated with LY5. After 30 days, imaging was performed to evaluate the effects of LY5 on the development of pulmonary metastasis.

    Article Snippet: To investigate the pharmacodynamics (PD) effects of LY5, pSTAT3 (p-Tyr705, ab76315, Abcam) and total STAT3 (ab119352, Abcam) were evaluated in pulmonary metastatic lesions using IHC in lung samples from mice euthanized 24 hrs after their last dose.

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Western Blot, Inhibition, Stable Transfection, Expressing, Luciferase, Injection, Imaging

    LY5 inhibits pSTAT3 in sarcoma cell lines. (A) The tumor cell lines RH30, EW8, and RD were treated with increasing concentrations of LY5 for 30 minutes. Cell lysates were collected for Western blotting of p-STAT3, total STAT3, and GAPDH. GAPDH was used as loading control. (B-C) The SJSA cell line was serum-starved overnight and then left untreated or treated with LY5 for two hours followed by stimulation with 50 ng/mL of OSM, IL-6, IFN-γ, IFN-α, or IL-4 for 30 minutes prior to collection of cell lysates for Western blotting of p-STAT3, total STAT3 p-STAT1, total STAT1, p-STAT2, p-STAT4, or p-STAT6. GAPDH was used as loading control.

    Journal: PLoS ONE

    Article Title: Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma

    doi: 10.1371/journal.pone.0181885

    Figure Lengend Snippet: LY5 inhibits pSTAT3 in sarcoma cell lines. (A) The tumor cell lines RH30, EW8, and RD were treated with increasing concentrations of LY5 for 30 minutes. Cell lysates were collected for Western blotting of p-STAT3, total STAT3, and GAPDH. GAPDH was used as loading control. (B-C) The SJSA cell line was serum-starved overnight and then left untreated or treated with LY5 for two hours followed by stimulation with 50 ng/mL of OSM, IL-6, IFN-γ, IFN-α, or IL-4 for 30 minutes prior to collection of cell lysates for Western blotting of p-STAT3, total STAT3 p-STAT1, total STAT1, p-STAT2, p-STAT4, or p-STAT6. GAPDH was used as loading control.

    Article Snippet: To investigate the pharmacodynamics (PD) effects of LY5, pSTAT3 (p-Tyr705, ab76315, Abcam) and total STAT3 (ab119352, Abcam) were evaluated in pulmonary metastatic lesions using IHC in lung samples from mice euthanized 24 hrs after their last dose.

    Techniques: Western Blot

    piR-54265 interacts with PIWIL2, facilitating PIWIL2/STAT3/p-SRC formation. (A) RNA immunoprecipitation assays showed specific bind of piR-54265 to PIWIL2. Results are mean ± SEM of piR-54265 enrichment relative to input from three independent experiments. (B) Biotin-labeled piR-54265 RNA pulldown coupled with western blot analysis revealed interaction of piR-54265 with PIWIL2, STAT3 and p-SRC. (C) Schematic of the domain structure of PIWIL2 protein. (D) Biotin-labeled piR-54265 RNA pulldown from lysates containing FLAG-tagged full-length or truncated PIWIL2 protein coupled with Western blot analysis revealed the interaction of piR-54265 with PIWIL2 via the PIWI domain in PIWIL2. (E) Co-immunoprecipitation assays revealed the interaction of STAT3 with PIWIL2 via the PAZ domain in PIWIL2. (F) RNA immunoprecipitation assays showed specific association of piR-54265 with STAT3 and p-SRC in CRC cells. Results are mean ± SEM of piR-54265 enrichment relative to input from three independent experiments. (G) Reciprocal immunoprecipitation assays showed that the interaction among PIWIL2, STAT3 and SRC in CRC cells was affected by piR-54265 expression. (H) Effects of overexpression or knockdown of piR-54265 on the expression levels of STAT3 and SRC and their phosphorylated forms detected by western blot.

    Journal: Theranostics

    Article Title: PIWI-interacting RNA-54265 is oncogenic and a potential therapeutic target in colorectal adenocarcinoma

    doi: 10.7150/thno.28001

    Figure Lengend Snippet: piR-54265 interacts with PIWIL2, facilitating PIWIL2/STAT3/p-SRC formation. (A) RNA immunoprecipitation assays showed specific bind of piR-54265 to PIWIL2. Results are mean ± SEM of piR-54265 enrichment relative to input from three independent experiments. (B) Biotin-labeled piR-54265 RNA pulldown coupled with western blot analysis revealed interaction of piR-54265 with PIWIL2, STAT3 and p-SRC. (C) Schematic of the domain structure of PIWIL2 protein. (D) Biotin-labeled piR-54265 RNA pulldown from lysates containing FLAG-tagged full-length or truncated PIWIL2 protein coupled with Western blot analysis revealed the interaction of piR-54265 with PIWIL2 via the PIWI domain in PIWIL2. (E) Co-immunoprecipitation assays revealed the interaction of STAT3 with PIWIL2 via the PAZ domain in PIWIL2. (F) RNA immunoprecipitation assays showed specific association of piR-54265 with STAT3 and p-SRC in CRC cells. Results are mean ± SEM of piR-54265 enrichment relative to input from three independent experiments. (G) Reciprocal immunoprecipitation assays showed that the interaction among PIWIL2, STAT3 and SRC in CRC cells was affected by piR-54265 expression. (H) Effects of overexpression or knockdown of piR-54265 on the expression levels of STAT3 and SRC and their phosphorylated forms detected by western blot.

    Article Snippet: Antibodies against STAT3 (ab119352), p-STAT3 (p-Y705, ab76315), SRC (ab109381), BCL-XL (ab32370), active CASPASE-3 (ab32351), MMP2 (ab86607) or MMP9 (ab58803) were from Abcam.

    Techniques: Immunoprecipitation, Labeling, Western Blot, Expressing, Over Expression

    piR-54265 enhances oncogenic STAT3 signaling. (A) Effects of piR-54265 on expression and/or activation of proliferation and metastasis-related STAT3 downstream modules in CRC cells detected by western blot. (B-C) Immunohistochemical (IHC) staining of proliferation- and metastasis-related STAT3 downstream modules in mouse xenograft tumors of CRC cells with overexpression or knockdown of piR-54265. IHC staining of proliferation-related molecules (B) and metastasis-related molecules (C). 200×; scale bars, 50 μm. (D) Knockdown of PIWIL2 or STAT3 affects piR-54265-induced CRC cell proliferation (each point in the curve represents mean ± SEM; N=6; **, P

    Journal: Theranostics

    Article Title: PIWI-interacting RNA-54265 is oncogenic and a potential therapeutic target in colorectal adenocarcinoma

    doi: 10.7150/thno.28001

    Figure Lengend Snippet: piR-54265 enhances oncogenic STAT3 signaling. (A) Effects of piR-54265 on expression and/or activation of proliferation and metastasis-related STAT3 downstream modules in CRC cells detected by western blot. (B-C) Immunohistochemical (IHC) staining of proliferation- and metastasis-related STAT3 downstream modules in mouse xenograft tumors of CRC cells with overexpression or knockdown of piR-54265. IHC staining of proliferation-related molecules (B) and metastasis-related molecules (C). 200×; scale bars, 50 μm. (D) Knockdown of PIWIL2 or STAT3 affects piR-54265-induced CRC cell proliferation (each point in the curve represents mean ± SEM; N=6; **, P

    Article Snippet: Antibodies against STAT3 (ab119352), p-STAT3 (p-Y705, ab76315), SRC (ab109381), BCL-XL (ab32370), active CASPASE-3 (ab32351), MMP2 (ab86607) or MMP9 (ab58803) were from Abcam.

    Techniques: Expressing, Activation Assay, Western Blot, Immunohistochemistry, Staining, Over Expression

    Leptin-driven internalization of GluA1 requires JAK2-STAT3 signaling. (A) Representative confocal images of surface GluA1 immunolabeling in 8–12 DIV hippocampal neurons in control conditions and after application of leptin (50 nM; 30 minutes), AG490 (10 μM), stattic (50 μM), and leptin plus AG490 or stattic. The ability of leptin to internalize GluA1 was reduced after JAK2-STAT3 inhibition. Scale bars represent 20 μM. (B) Histogram of pooled data illustrating the relative intensity of surface GluA1 in control conditions and after treatment with leptin, and in the combined presence of AG490 (10 μM), hexabromocyclohexane (50 μM), stattic (50 μM), and nifuroxazide (5 μM). Inhibition of JAK2-STAT3 signaling prevents leptin-driven GluA1 internalization in culture. (C) Representative confocal images of surface GluA1 (green) and p-JAK2 (red) in control and leptin-treated hippocampal neurons (8–12 DIV). “1”, “2”, and “3” are zoomed in dendritic regions of the representative images and show co-localization of surface GluA1 and p-JAK2 labeling. Application of leptin reduces surface GluA1 labeling and this is accompanied by increased p-JAK2 levels. (D) Histogram of pooled data showing the relative intensity of surface GluA1 (filled bars) and p-JAK2 (open bars) labeling in control conditions and after leptin (50 nM; 30 minutes), D-AP5 (50 μM), and SD-1008 (10 μM) treatment or in the presence of leptin plus D-AP5 or SD-1008. (E) Representative confocal images of surface GluA1 (green) and p-STAT3 (red) in control and leptin-treated neurons (8–12 DIV). “1”, “2”, and “3” are zoomed in dendritic regions of the representative images and show co-localization of surface GluA1 and p-STAT3 labeling. Leptin reduces GluA1 surface expression and this is accompanied by an increase in p-STAT3. (F) Histogram of pooled data indicating the relative intensity of surface GluA1 (filled bars) and p-STAT3 (open bars) labeling in control conditions, after leptin (50 nM; 30 minutes), D-AP5 (50 μM), and SD-1008 (10 μM) treatment or in the presence of leptin plus D-AP5 or SD-1008. The leptin-dependent effects on GluA1 trafficking are accompanied by an increase in JAK2 and STAT3 activity. In this and subsequent figures, *, **, and *** represent p

    Journal: Neurobiology of Aging

    Article Title: Age-dependent regulation of excitatory synaptic transmission at hippocampal temporoammonic-CA1 synapses by leptin

    doi: 10.1016/j.neurobiolaging.2018.05.007

    Figure Lengend Snippet: Leptin-driven internalization of GluA1 requires JAK2-STAT3 signaling. (A) Representative confocal images of surface GluA1 immunolabeling in 8–12 DIV hippocampal neurons in control conditions and after application of leptin (50 nM; 30 minutes), AG490 (10 μM), stattic (50 μM), and leptin plus AG490 or stattic. The ability of leptin to internalize GluA1 was reduced after JAK2-STAT3 inhibition. Scale bars represent 20 μM. (B) Histogram of pooled data illustrating the relative intensity of surface GluA1 in control conditions and after treatment with leptin, and in the combined presence of AG490 (10 μM), hexabromocyclohexane (50 μM), stattic (50 μM), and nifuroxazide (5 μM). Inhibition of JAK2-STAT3 signaling prevents leptin-driven GluA1 internalization in culture. (C) Representative confocal images of surface GluA1 (green) and p-JAK2 (red) in control and leptin-treated hippocampal neurons (8–12 DIV). “1”, “2”, and “3” are zoomed in dendritic regions of the representative images and show co-localization of surface GluA1 and p-JAK2 labeling. Application of leptin reduces surface GluA1 labeling and this is accompanied by increased p-JAK2 levels. (D) Histogram of pooled data showing the relative intensity of surface GluA1 (filled bars) and p-JAK2 (open bars) labeling in control conditions and after leptin (50 nM; 30 minutes), D-AP5 (50 μM), and SD-1008 (10 μM) treatment or in the presence of leptin plus D-AP5 or SD-1008. (E) Representative confocal images of surface GluA1 (green) and p-STAT3 (red) in control and leptin-treated neurons (8–12 DIV). “1”, “2”, and “3” are zoomed in dendritic regions of the representative images and show co-localization of surface GluA1 and p-STAT3 labeling. Leptin reduces GluA1 surface expression and this is accompanied by an increase in p-STAT3. (F) Histogram of pooled data indicating the relative intensity of surface GluA1 (filled bars) and p-STAT3 (open bars) labeling in control conditions, after leptin (50 nM; 30 minutes), D-AP5 (50 μM), and SD-1008 (10 μM) treatment or in the presence of leptin plus D-AP5 or SD-1008. The leptin-dependent effects on GluA1 trafficking are accompanied by an increase in JAK2 and STAT3 activity. In this and subsequent figures, *, **, and *** represent p

    Article Snippet: A second primary antibody was then applied to compare GluA1 surface immunostaining relative to p-JAK2 (rabbit anti-JAK2 phospho Y1007-1008; 1:250; Abcam, UK), p-STAT3 (rabbit anti-STAT3 phospho Y705; 1:500; Abcam, UK), or PSD-95 (mouse anti-PSD-95; 1:500; Thermo Fisher, UK). p-JAK2 and p-STAT3 staining was visualized by addition of an anti-rabbit Alexa 555–conjugated secondary antibody (1:250; Life Technologies, UK) for 30 minutes, whereas an Alexa 569–conjugated anti-mouse secondary antibody (1:200; Thermo Fisher, UK) was used to visualize PSD-95 labeling ( ).

    Techniques: Immunolabeling, Inhibition, Labeling, Expressing, Activity Assay

    Leptin-induced LTD requires STAT3-dependent gene transcription. (A–F) Plots of pooled and normalized data illustrating the effects of leptin (25 nM) on excitatory synaptic transmission in the presence of various inhibitors. In this and subsequent figures, each point is the average of four successive responses and representative synaptic traces for each experiment are shown above each plot and for the time indicated. Treatment with the STAT3 inhibitor, stattic (50 μM; [A]); the JAK-STAT3 inhibitor, nifuroxazide (5 μM; [B]); or the JAK2-STAT3 inhibitor, SD-1008 (10 μM; [C]), all inhibited leptin-induced LTD, indicating involvement of STAT3. Likewise, inhibition of STAT3 binding to DNA (galiellalactone; 10 μM; [D]), nuclear export (leptomycin B; 50 nM; [E]), or gene transcription (actinomycin D; 25 μM; [F]) also inhibited leptin-induced LTD. Thus leptin-induced LTD involves JAK2-STAT3 signaling and subsequent gene transcription. Abbreviations: LTD, long-term depression.

    Journal: Neurobiology of Aging

    Article Title: Age-dependent regulation of excitatory synaptic transmission at hippocampal temporoammonic-CA1 synapses by leptin

    doi: 10.1016/j.neurobiolaging.2018.05.007

    Figure Lengend Snippet: Leptin-induced LTD requires STAT3-dependent gene transcription. (A–F) Plots of pooled and normalized data illustrating the effects of leptin (25 nM) on excitatory synaptic transmission in the presence of various inhibitors. In this and subsequent figures, each point is the average of four successive responses and representative synaptic traces for each experiment are shown above each plot and for the time indicated. Treatment with the STAT3 inhibitor, stattic (50 μM; [A]); the JAK-STAT3 inhibitor, nifuroxazide (5 μM; [B]); or the JAK2-STAT3 inhibitor, SD-1008 (10 μM; [C]), all inhibited leptin-induced LTD, indicating involvement of STAT3. Likewise, inhibition of STAT3 binding to DNA (galiellalactone; 10 μM; [D]), nuclear export (leptomycin B; 50 nM; [E]), or gene transcription (actinomycin D; 25 μM; [F]) also inhibited leptin-induced LTD. Thus leptin-induced LTD involves JAK2-STAT3 signaling and subsequent gene transcription. Abbreviations: LTD, long-term depression.

    Article Snippet: A second primary antibody was then applied to compare GluA1 surface immunostaining relative to p-JAK2 (rabbit anti-JAK2 phospho Y1007-1008; 1:250; Abcam, UK), p-STAT3 (rabbit anti-STAT3 phospho Y705; 1:500; Abcam, UK), or PSD-95 (mouse anti-PSD-95; 1:500; Thermo Fisher, UK). p-JAK2 and p-STAT3 staining was visualized by addition of an anti-rabbit Alexa 555–conjugated secondary antibody (1:250; Life Technologies, UK) for 30 minutes, whereas an Alexa 569–conjugated anti-mouse secondary antibody (1:200; Thermo Fisher, UK) was used to visualize PSD-95 labeling ( ).

    Techniques: Transmission Assay, Inhibition, Binding Assay

    GFs-HP hydrogel treatment activates SCs proliferation through MAPK/ERK, PI3K/Akt and JAK/STAT3 pathways. A. Immunoblot for p -AKT, p -ERK, p -STAT3; total proteins amount of AKT, ERK, STAT3 served as loading control; B–D: Densitometric quantification of p -AKT/AKT, p -ERK/ERK and p -STAT3/STAT3, respectively. GAPDH was used for band density normalization. Data presented as Mean±SEM n=5 for each group. Significance markers: Free GFs vs PNI-diabetes: * P

    Journal: Biomaterials

    Article Title: Heparin-Poloxamer Thermosensitive Hydrogel Loaded with bFGF and NGF Enhances Peripheral Nerve Regeneration in Diabetic Rats

    doi: 10.1016/j.biomaterials.2018.03.044

    Figure Lengend Snippet: GFs-HP hydrogel treatment activates SCs proliferation through MAPK/ERK, PI3K/Akt and JAK/STAT3 pathways. A. Immunoblot for p -AKT, p -ERK, p -STAT3; total proteins amount of AKT, ERK, STAT3 served as loading control; B–D: Densitometric quantification of p -AKT/AKT, p -ERK/ERK and p -STAT3/STAT3, respectively. GAPDH was used for band density normalization. Data presented as Mean±SEM n=5 for each group. Significance markers: Free GFs vs PNI-diabetes: * P

    Article Snippet: The following primary antibodies were used: Ace-tubulin (1μg/ml, T7451, Sigma), Tyr-tubulin (1μg/ml, ab6046, Abcam), Tau (1μg/ml, ab18207, Abcam), GAP43 (0.67μg/ml, sc-17790, Santa), PCNA (0.67μg/ml, sc-25280, Santa), Ki67 (1μg/ml, ab15580, Abcam), p -AKT (1μg/ml, ab183758, Abcam), AKT (0.67μg/ml, sc-8312, Santa), p -ERK (0.67μg/ml, sc-7383, Santa), ERK (0.67μg/ml, sc-292838, Santa), p -STAT3 (1μg/ml, ab76315, Abcam), STAT3 (1μg/ml, ab68153, Abcam) and GAPDH (0.1μg/ml, AP0063, Bioworld).

    Techniques: