phosphorylated (p-)jak2 antibody  (Cell Signaling Technology Inc)

Journal: Thoracic Cancer

Article Title: miR ‐224‐5p Suppresses Non‐Small Cell Lung Cancer via IL6ST ‐Mediated Regulation of the JAK2 / STAT3 Pathway

doi: 10.1111/1759-7714.15516

Figure Lengend Snippet: miR‐224‐5p inhibits IL6ST transcription and inactivates the JAK2/STAT3 signaling pathway. (A) Venn diagram showing statistical analysis of miR‐224‐5p–related targets predicted by TargetScan, PITA, and miRanda database. (B) Correlation between IL6ST expression and miR‐224‐5p expression based on database analysis. (C) Survival curve analysis of IL6ST expression in NSCLC using the GEPIA2 database. (D) Prediction of IL6ST binding sites with miR‐224‐5p by database analysis. (E) Luciferase gene reporter assay analysis of the binding of miR‐224‐5p with IL6ST 3′‐UTR fluorescent plasmid and mutant plasmid in H226 cells. (F) RT‐qPCR detection of IL6ST mRNA levels after stable introduction of miR‐224‐5p mimic in H226 cells. (G) Western blot analysis was used to assess the expression levels of IL6ST, JAK2, p‐JAK2, STAT3, and p‐STAT3 proteins in A549 cells, A549 stably introduced with miR‐224‐5p inhibitor, H226 cells, and H226 cells introduced with miR‐224‐5p mimic. (H) Immunofluorescence analysis of the phosphorylated JAK and STAT3 expression and the nuclear localization of p‐STAT3. (H) Immunofluorescence analysis of the phosphorylated JAK and STAT3 expression and the nuclear localization of p‐STAT3. The statistical significance was expressed as * p < 0.05; ** p < 0.01; and *** p < 0.001. Values are expressed as mean ± standard error.

Article Snippet: The normalized proteins were separated by SDS‐PAGE, incubating with primary antibodies against IL6ST (Affinity, cat#AF6291), phosphorylated JAK2 (p‐JAK2) (Tyr1007/1008) (CST, #3776), JAK2 (Affinity, cat# AF6022), phosphorylated STAT3 (p‐STAT3) (Tyr705) (CST, #4113), STAT3 (Affinity, cat# AF6294), and tubulin (Affinity, cat# AF7011) at 4°C overnight.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Western Blot, Stable Transfection, Immunofluorescence

Journal: Thoracic Cancer

Article Title: miR ‐224‐5p Suppresses Non‐Small Cell Lung Cancer via IL6ST ‐Mediated Regulation of the JAK2 / STAT3 Pathway

doi: 10.1111/1759-7714.15516

Figure Lengend Snippet: IL6ST overexpression reverses the inhibition of miR‐224‐5p and activation of the JAK2/STAT3 pathway in NSCLC. (A) IL6ST expression levels were detected by western blotting after H226 cells were transfected with IL6ST overexpression plasmid for 48 h. (B) The expression levels of IL6ST, JAK2, STAT3 proteins, and phosphorylated JAK2 and STAT3 proteins were assessed by western blotting in H226 cells under three conditions: overexpression of IL6ST, introduction of miR‐224‐5p mimic, and overexpression of IL6ST followed by introduction of miR‐224‐5p mimic. (C) Detecting the expression levels of phosphorylated JAK2 and STAT3 proteins and the nuclear localization of phosphorylated STAT3 under different cell conditions using an immunofluorescence assay. (D) Evaluation of the difference in the invasion level of H226 cells under different conditions by the Transwell assay. (E) The difference in the migration ability of the four cell types at 0, 24, and 48 h using a wound healing assay. The statistical significance was expressed as * p < 0.05; ** p < 0.01; and *** p < 0.001. Values are expressed as mean ± standard error.

Article Snippet: The normalized proteins were separated by SDS‐PAGE, incubating with primary antibodies against IL6ST (Affinity, cat#AF6291), phosphorylated JAK2 (p‐JAK2) (Tyr1007/1008) (CST, #3776), JAK2 (Affinity, cat# AF6022), phosphorylated STAT3 (p‐STAT3) (Tyr705) (CST, #4113), STAT3 (Affinity, cat# AF6294), and tubulin (Affinity, cat# AF7011) at 4°C overnight.

Techniques: Over Expression, Inhibition, Activation Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Immunofluorescence, Transwell Assay, Migration, Wound Healing Assay

Journal: International Journal of Molecular Medicine

Article Title: SPP1 promotes the polarization of M2 macrophages through the Jak2/Stat3 signaling pathway and accelerates the progression of idiopathic pulmonary fibrosis

doi: 10.3892/ijmm.2024.5413

Figure Lengend Snippet: SPP1 induces macrophage M2 polarization through the JAK2/STAT3 pathway. (A and B) After treating PBMC-m and THP-1 cells with PBS, BLM, and BLM + si_SPP1 for 48 h, the protein expression of SPP1, JAK2, STAT3, p-JAK2 and p-STAT3 were detected (with the experiment repeated three times). (C-F) PBS, BLM and BLM + Stattic were used to treat PBMC-m and THP-1 cells, and the supernatants were collected and co-cultured with BEAS-2B and WI-38 cells, respectively, for cell scratch assays (magnification, ×100) (with the experiment repeated three times). Data are presented as the mean ± standard deviation. ** P<0.01 and *** P<0.001. SPP1, secreted phosphoprotein 1; BLM, bleomycin. PBMC, peripheral blood mononuclear cells; p-, phosphorylated; ns, not significant.

Article Snippet: The antibodies used in the experiment included: SPP1 (Proteintech Group, Inc.; cat. no. 22952-1-AP; 1:2,000), COL3A1 (Proteintech Group, Inc.; cat. no. 68320-1-Ig; 1:5,000), E-cadherin (Cell Signaling Technology, Inc.; cat. no. 3195T; 1:1,000), N-cadherin (Cell Signaling Technology, Inc.; cat. no. 4061T; 1:1,000), Vimentin (Cell Signaling Technology, Inc.; cat. no. 5741T; 1:1,000), phosphorylated (p)-JAK2 (ABclonal Biotech Co., Ltd.; cat. no. AP0531; 1:1,000), p-STAT3 (ABclonal Biotech Co., Ltd.; cat. no. AP0705; 1:1,000), JAK2 (ABclonal Biotech Co., Ltd.; cat. no. A11497; 1:1,000), STAT3 (ABclonal Biotech Co., Ltd.; cat. no. A1192; 1:1,000), GAPDH (ABclonal Biotech Co., Ltd.; cat. no. AC002; 1:5,000) and HRP-conjugated secondary antibody (ABclonal Biotech Co., Ltd.; cat. no. AS063; 1:10,000).

Techniques: Expressing, Cell Culture, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: SPP1 promotes the polarization of M2 macrophages through the Jak2/Stat3 signaling pathway and accelerates the progression of idiopathic pulmonary fibrosis

doi: 10.3892/ijmm.2024.5413

Figure Lengend Snippet: In vivo inhibition of SPP1 expression can effectively treat IPF in mice. (A) Schematic illustration of the SPP1 inhibitor treatment in an IPF mouse model. (B) Graphical representation of weight changes in three groups of mice. (C) Images of lung tissues from three groups of mice at the end of the experiment (scale bar, 1 cm). (D) Analysis of H&E, Masson and Sirius Red staining of lung tissues from three groups of mice (each dot represents one mouse; scale bar, 200 μ m). (E) Immunofluorescence detection of FAM13A expression in mouse lung tissues (red) (scale bar, 50 μ m). (F) Immunofluorescence analysis of SPP1 (red) expression and localization in lung tissues of normal, IPF and SPP1-inhibitor treated IPF mice, with macrophages (F4/80, green) and cell nuclei (DAPI, blue) (scale bar, 5 μ m). (G) Detection of p-JAK2 (green) and p-STAT3 (green) expression in lung tissues from normal, IPF and SPP1 inhibitor-treated IPF mice, with macrophages (F4/80, red), cell nuclei (DAPI, blue) (scale bar, 5 μ m). Data are presented as the mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001. SPP1, secreted phosphoprotein 1; IPF, idiopathic pulmonary fibrosis; p-, phosphorylated; BLM, bleomycin; ns, not significant.

Article Snippet: The antibodies used in the experiment included: SPP1 (Proteintech Group, Inc.; cat. no. 22952-1-AP; 1:2,000), COL3A1 (Proteintech Group, Inc.; cat. no. 68320-1-Ig; 1:5,000), E-cadherin (Cell Signaling Technology, Inc.; cat. no. 3195T; 1:1,000), N-cadherin (Cell Signaling Technology, Inc.; cat. no. 4061T; 1:1,000), Vimentin (Cell Signaling Technology, Inc.; cat. no. 5741T; 1:1,000), phosphorylated (p)-JAK2 (ABclonal Biotech Co., Ltd.; cat. no. AP0531; 1:1,000), p-STAT3 (ABclonal Biotech Co., Ltd.; cat. no. AP0705; 1:1,000), JAK2 (ABclonal Biotech Co., Ltd.; cat. no. A11497; 1:1,000), STAT3 (ABclonal Biotech Co., Ltd.; cat. no. A1192; 1:1,000), GAPDH (ABclonal Biotech Co., Ltd.; cat. no. AC002; 1:5,000) and HRP-conjugated secondary antibody (ABclonal Biotech Co., Ltd.; cat. no. AS063; 1:10,000).

Techniques: In Vivo, Inhibition, Expressing, Staining, Immunofluorescence, Standard Deviation

Journal: Renal Failure

Article Title: M2 macrophage infusion ameliorates diabetic glomerulopathy via the JAK2/STAT3 pathway in db/db mice

doi: 10.1080/0886022X.2024.2378210

Figure Lengend Snippet: M2 macrophage infusion downregulated JAK2/STAT3 expression. (A) Immunoblot analysis of mouse kidneys from the normal group, DN group, and M2 group. (B,C) Relative protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in the kidney are shown. The relative protein levels were quantified as the ratio of p-JAK2 to JAK2 and that of p-STAT3 to STAT3. (D) Immunoblot analysis of SV40-MES-13 cells cultured alone (con group), stimulated with 30 mmol/L glucose (HG group), or stimulated with high glucose (M2 group) to detect M2 macrophages. (E,F) The relative protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in vitro are shown. The relative protein level was quantified by determining the ratio of p-JAK2 to JAK2 and that of p-STAT3 to STAT3. The values are the means ± SD s of three individual experiments. ** p < 0.01.

Article Snippet: The following antibodies were used: total or phosphorylated JAK2 (p-JAK2) (3230S and 3776S, 1:1000; rabbit, Cell Signaling Technology, MA, USA); total or phosphorylated STAT3 (p-STAT3) (9139S, 1:1000; mouse, Cell Signaling Technology, MA, USA; 9145S, 1:2000; rabbit, Cell Signaling Technology, MA, USA); β-tubulin (TA-10, 1:2000; mouse, ZSGB-Bio, China); and β-actin (TA-09, 1:2000; mouse, ZSGB-Bio, China).

Techniques: Expressing, Western Blot, Cell Culture, In Vitro

Journal: Renal Failure

Article Title: M2 macrophage infusion ameliorates diabetic glomerulopathy via the JAK2/STAT3 pathway in db/db mice

doi: 10.1080/0886022X.2024.2378210

Figure Lengend Snippet: The effects of M2 macrophages were mediated by the downregulation of JAK2/STAT3. (A) and (B) Immunofluorescence analysis of Col-4 + and fibronectin + SV40-MES-13 cells cultured alone, stimulated with 30 mmol/L glucose or stimulated with high glucose in the presence of M2 macrophages transfected with the JAK2 NC or the JAK2 mimic. Scale bar = 20 μm. The quantification of Col-4 + and fibronectin + cells is presented in (C,D). (E) Quantitative RT–PCR analysis of Col-4 and fibronectin expression in SV40-MES-13 cells. The results are presented relative to those of SV40-MES-13 cells cultured alone, which were set as 1. (F–H) The levels of MCP-1, IL-1β, IL-6, and TNF-α in the medium of SV40-MES-13 cells cultured alone, stimulated with 30 mmol/L glucose or stimulated with high glucose in the presence of M2 macrophages treated with the JAK2 NC or the JAK2 mimic. (I) Quantitative RT–PCR analysis of inflammatory factors in SV40-MES-13 cells in the four groups. The results are presented relative to those of the control group, which were set as 1. Results were determined by evaluating at least 8 random fields in each section. The values are the means ± SD s of three individual experiments. ** p < 0.01.

Article Snippet: The following antibodies were used: total or phosphorylated JAK2 (p-JAK2) (3230S and 3776S, 1:1000; rabbit, Cell Signaling Technology, MA, USA); total or phosphorylated STAT3 (p-STAT3) (9139S, 1:1000; mouse, Cell Signaling Technology, MA, USA; 9145S, 1:2000; rabbit, Cell Signaling Technology, MA, USA); β-tubulin (TA-10, 1:2000; mouse, ZSGB-Bio, China); and β-actin (TA-09, 1:2000; mouse, ZSGB-Bio, China).

Techniques: Immunofluorescence, Cell Culture, Transfection, Quantitative RT-PCR, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: BMSC‑derived exosome‑mediated miR‑25‑3p delivery protects against myocardial ischemia/reperfusion injury by constraining M1‑like macrophage polarization

doi: 10.3892/mmr.2024.13266

Figure Lengend Snippet: BMSC-Exo-25-3p inhibited I/R-induced inflammatory response in rats. Immunofluorescence staining of (A) iNOS and (B) CD163 of myocardial tissue (scale bar, 50 µm). Gene expression of proinflammatory cytokines (C) IL-1β and (D) IL-6 in myocardial tissue. Gene expression of anti-inflammatory cytokines (E) IL-10 and (F) Arg-1 in myocardial tissue. (G) Representative western blotting images of the JAK2/STAT3 signaling pathway. Quantitative analysis of (H) p-JAK2 and (I) p-STAT3. **P<0.01, *P<0.05 vs. Sham group. ## P<0.01, # P<0.05 vs. I/R + PBS group. All data were presented as mean ± SD. Exo, exosome; I/R, ischemia/reperfusion; BMSC, bone marrow mesenchymal stem cells; IL, interleukin; Arg-1, Arginase-1; JAK2, Janus kinase 2; p-, phosphorylated; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The primary antibodies used were against CD9 (1:500; cat. no. WL01236, Wanleibio Co., Ltd.), CD63 (1:1,000; cat. no. D111314-0100100; Sangon Biotech Co., Ltd.), JAK2 (1:1,000; cat. no. 3203; Cell Signaling Technology, Inc.), phosphorylated (p-)JAK2 (1:1,000; cat. no. 37745; Cell Signaling Technology, Inc.), STAT3 (1:2,000; cat. no. 9139; Cell Signaling Technology, Inc.), p-STAT3 (1:2,000; cat. no. 9145; Cell Signaling Technology, Inc.) and GAPDH (1:5,000; cat. no. P07486; Sangon Biotech Co., Ltd.).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: BMSC‑derived exosome‑mediated miR‑25‑3p delivery protects against myocardial ischemia/reperfusion injury by constraining M1‑like macrophage polarization

doi: 10.3892/mmr.2024.13266

Figure Lengend Snippet: BMSC-Exo-25-3p alleviated H/R-induced injury in H9C2 cells. (A) Phagocytosis of Dil-labeled BMSC-Exo in H9C2 cells observed by a confocal microscope (scale bar, 50 µm). (B) Expression of miR-25-3p in H9C2 cells after co-incubation assessed by RT-qPCR. (C) Expression of miR-25-3p in H/R-induced injury in H9C2 cells. (D) LDH and (E) CK activity in cell culture supernatant. (F) Representative western blotting images of the JAK2/STAT3 signaling pathway. Quantity analysis of (G) p-JAK2 and (H) p-STAT3 in H9C2 cells. **P<0.01, *P<0.05 vs. control group. ## P<0.01, # P<0.05 vs. H/R + PBS group. ▽ P<0.05 vs. H/R + BMSC-Exo group. & P<0.01 vs. PBS group, BMSC-Exo group or BMSC-Exo-NC group. All data were presented as mean ± SD. Exo, exosome; BMSC, bone marrow mesenchymal stem cells; NC, negative control; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; CK, creatine kinase; JAK2, Janus kinase 2; p-, phosphorylated; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The primary antibodies used were against CD9 (1:500; cat. no. WL01236, Wanleibio Co., Ltd.), CD63 (1:1,000; cat. no. D111314-0100100; Sangon Biotech Co., Ltd.), JAK2 (1:1,000; cat. no. 3203; Cell Signaling Technology, Inc.), phosphorylated (p-)JAK2 (1:1,000; cat. no. 37745; Cell Signaling Technology, Inc.), STAT3 (1:2,000; cat. no. 9139; Cell Signaling Technology, Inc.), p-STAT3 (1:2,000; cat. no. 9145; Cell Signaling Technology, Inc.) and GAPDH (1:5,000; cat. no. P07486; Sangon Biotech Co., Ltd.).

Techniques: Labeling, Microscopy, Expressing, Incubation, Quantitative RT-PCR, Activity Assay, Cell Culture, Western Blot, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: BMSC‑derived exosome‑mediated miR‑25‑3p delivery protects against myocardial ischemia/reperfusion injury by constraining M1‑like macrophage polarization

doi: 10.3892/mmr.2024.13266

Figure Lengend Snippet: LPS-stimulated RAW 264.7 macrophage cocultured with H/R-induced H9C2 cells. (A) Representative western blotting images of the JAK2/STAT3 signaling pathway in LPS-stimulated RAW 264.7 cells. Quantitative analysis of (B) p-JAK2 and (C) p-STAT3. (D) LDH and (E) CK activity in cell culture supernatant of H9C2 cells pretreated with CM obtained from LPS-stimulated RAW 264.7 macrophages. **P<0.01, *P<0.05 vs. Control group. ## P<0.01, # P<0.05 vs. LPS + PBS group. ▽ P<0.05 vs LPS + BMSC-Exo group. && P<0.01, & P<0.05 vs. H/R + CM1 group. All data were presented as mean ± SD. Exo, exosome; BMSC, bone marrow mesenchymal stem cells; LPS, lipopolysaccharide; JAK2, Janus kinase 2; p-, phosphorylated; STAT3, signal transducer and activator of transcription 3; LDH, lactate dehydrogenase; CK, creatine kinase; CM, conditioned medium.

Article Snippet: The primary antibodies used were against CD9 (1:500; cat. no. WL01236, Wanleibio Co., Ltd.), CD63 (1:1,000; cat. no. D111314-0100100; Sangon Biotech Co., Ltd.), JAK2 (1:1,000; cat. no. 3203; Cell Signaling Technology, Inc.), phosphorylated (p-)JAK2 (1:1,000; cat. no. 37745; Cell Signaling Technology, Inc.), STAT3 (1:2,000; cat. no. 9139; Cell Signaling Technology, Inc.), p-STAT3 (1:2,000; cat. no. 9145; Cell Signaling Technology, Inc.) and GAPDH (1:5,000; cat. no. P07486; Sangon Biotech Co., Ltd.).

Techniques: Western Blot, Activity Assay, Cell Culture, Control