anti phospho stat3  (Bio-Techne corporation)

Journal: The FASEB Journal

Article Title: CSPG4 Mediates Inflammatory, Cell Death, and Senescence Responses in Enteric Glia Exposed to Clostridioides difficile Toxins

doi: 10.1096/fj.202600333R

Figure Lengend Snippet: CSPG4 blockade reduces TcdB‐induced pSTAT3 nuclear translocation in enteric glial cells. (A) Representative photomicrographs of pSTAT3 (green) immunostaining, phalloidin (red) as a cytoplasm staining, and DAPI (blue) as a nuclear staining in enteric glial cells exposed to TcdA (50 ng/mL) or TcdB (1 ng/mL) at 18 h incubation in the presence of isotype control or anti‐CSPG4 antibodies (0.001 μg/mL). Scale bars: 50 μm. (B) Percentage of enteric glial cells ( n = 5, mean ± SEM) with positive nuclear pSTAT3 staining at 18 h of incubation with TcdA and TcdB in the presence of isotype control or anti‐CSPG4 antibodies (0.001 μg/mL). One‐way ANOVA followed by the Tukey test.

Article Snippet: After washing, cells were incubated overnight at 4°C with primary antibodies against CSPG4 (Abcam, ab275024, 1:400), nectin‐3 (PVRL3, Invitrogen, PA5‐51095, 1:200), LRP1 (Abcam, ab92544, 1:100), NFκB p65 (Santa Cruz Biotechnology, sc‐372, 1:100), or pSTAT3 (R&D Systems, AF4607, 1:100).

Techniques: Translocation Assay, Immunostaining, Staining, Incubation, Control

Journal: Translational Oncology

Article Title: Erianin ameliorates morphine tolerance and glioma progression through the JAK2-STAT3 pathway

doi: 10.1016/j.tranon.2025.102551

Figure Lengend Snippet: Erianin suppresses the JAK2/STAT3 signaling pathway in DRG after chronic morphine treatment. (A and B) JAK2 expression was detected in DRG of model mice. (C) JAK2 mRNA level was measured in spinal cord of model mice. (D and E) JAK2 expression was examined in model mice with or without Erianin treatment. * P < 0.05, ** P < 0.01 vs control group, ## P < 0.01 vs Morphine+NC group.

Article Snippet: Membranes were blocked with 5 % non-fat milk and incubated with primary antibodies against BDNF (Cat # 108,319, Abcam, 1:3000), JAK2 (Cat # 108,596, Abcam, 1:2000), p-STAT3 (Cat # AF4607, R&D systems, 1:1500), and GAPDH (Cat # 8245, Abcam, 1:10,000) overnight at 4 °C.

Techniques: Expressing, Control

Journal: Translational Oncology

Article Title: Erianin ameliorates morphine tolerance and glioma progression through the JAK2-STAT3 pathway

doi: 10.1016/j.tranon.2025.102551

Figure Lengend Snippet: Erianin upregulates miR-375 and miR-20a level and thus suppresses the JAK2/STAT3 signaling pathway in the DRG of glioma mice after chronic morphine treatment. (A) The levels of several miRNAs as indicated were measured in model mice with or without Erianin treatment via qPCR assays. (B) MiR-375 level was measured in DRG in model mice via qPCR assays. (C) MiR-20a level was examined in DRG of model mice via qPCR assays. (D) MiR-375 level was detected in spinal cord of model mice via qPCR assays. (E) MiR-20a level was examined in spinal cord of model mice. via qPCR assays (F) The correlation between JAK2 and miR-375 expression was measured in DRG of model mice via qPCR assays. (G) The correlation between JAK2 and miR-20a expression was determined in DRG of model mice via qPCR assays. (H) The protein levels of JAK2 and p-STAT3 were assessed in DRG of model mice with Erianin treatment as well as miRNA antagomir or not via western blot analysis. ** P < 0.01 vs control group, ns means no significance.

Article Snippet: Membranes were blocked with 5 % non-fat milk and incubated with primary antibodies against BDNF (Cat # 108,319, Abcam, 1:3000), JAK2 (Cat # 108,596, Abcam, 1:2000), p-STAT3 (Cat # AF4607, R&D systems, 1:1500), and GAPDH (Cat # 8245, Abcam, 1:10,000) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Translational Oncology

Article Title: Erianin ameliorates morphine tolerance and glioma progression through the JAK2-STAT3 pathway

doi: 10.1016/j.tranon.2025.102551

Figure Lengend Snippet: Erianin shows significant effectiveness in alleviating morphine tolerance in glioma-bearing mice through downregulation of BDNF expression and suppression of the JAK2/STAT3 signaling pathway, mediated by the upregulation of miR-375 and miR-20a.

Article Snippet: Membranes were blocked with 5 % non-fat milk and incubated with primary antibodies against BDNF (Cat # 108,319, Abcam, 1:3000), JAK2 (Cat # 108,596, Abcam, 1:2000), p-STAT3 (Cat # AF4607, R&D systems, 1:1500), and GAPDH (Cat # 8245, Abcam, 1:10,000) overnight at 4 °C.

Techniques: Expressing

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 (pSTAT3), AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Phospho-proteomics, Protein-Protein interactions, Ligation

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: In A) Sk-BR-3 human HER2-positive breast-carcinoma cells were stimulated with epidermal growth factor (EGF; 50 ng/ml) for 60 min, or left either serum-starved (Starvation control) or in complete-media control conditions; whole-cell lysate from K562 cells was loaded as a positive control in the right-most lane. Immunoblots were probed sequentially for STAT3 (≈70 kDa; Abcam, ab171359, 1:2 000), STAT5A (≈80 kDa; Abcam, ab213219, 1:1 000), ERK2 (≈42 kDa; Thermo Fisher, PA5-29636, 1:1 000), MEK1 (≈45 kDa; Abcam, ab239802, 1:1 000), AKT2 (≈55 kDa; Thermo Fisher, PA5-85518, 1:1 000), phospho-STAT3-Tyr705 (pSTAT3, ≈90–115 kDa; R&D, AF4607, 1:2 000) and pan-AKT (≈60 kDa; CST, #4691, 1:1 000). Membranes were finally reprobed for GAPDH (≈37 kDa; CST, #14C10, 1:1 000) and vinculin (≈120 kDa; CST, #E1E9V, 1:1 000) as loading controls. Molecular-weight markers are indicated on the left. For all panels, proteins were resolved on Bis-Tris SDS–PAGE gels (4–12 % or 10 % as specified in Methods) using MES or MOPS running buffer, transferred to PVDF, and detected by near-infrared fluorescence on a LI-COR Odyssey® imager. In B) estimated relative band intensities of key signaling proteins in Sk-BR-3 and K562 cells are shown. Fluorescence-based Western blot images (Supp (A)) were used to visually estimate band intensities of STAT3, phospho-STAT3 (Tyr705), STAT5A, ERK2, MEK1, AKT2, and pan-AKT in Sk-BR-3 cells under three conditions: serum starvation, complete media, and 60 min EGF treatment, and in K562 cells as a positive control. Band intensities were approximated by visual comparison of signal brightness and normalized to the corresponding housekeeping protein (GAPDH or vinculin) within each lane. Normalized values were plotted as grouped bar graphs to highlight relative abundance patterns across conditions. This semi-quantitative representation supports qualitative comparison and was not derived from raw densitometry. validates the antibody specificity and establishes the baseline expression of PLA target proteins in Sk-BR-3 and K562 cells. Panel A displays the Western blot images of Sk-BR-3 cells subjected to three conditions: serum starvation, complete media (control), and EGF stimulation for 60 minutes. K562 cell lysate is included as a positive control. Panel B presents the quantified band intensities, normalised to the housekeeping proteins GAPDH or vinculin and expressed as percentages. In Sk-BR-3 cells, total STAT3, ERK2, MEK1, AKT2, and pan-AKT were consistently detected across all conditions. STAT3 levels remained stable, while phosphorylation at Tyr705 (pSTAT3) dropped to undetectable levels under serum starvation, rose slightly under media control, and reached higher levels after 60 min of EGF treatment. This trend suggests growth-factor-responsive STAT3 activation despite unchanged total STAT3 levels. MEK1 and AKT2 expression decreased mildly in media control compared to both EGF and starvation conditions, while ERK2 and pan-AKT remained constant. STAT5A was not detected in any Sk-BR3 condition. In contrast, the K562 lysate showed robust STAT5A expression, confirming its hematopoietic lineage specificity. STAT3 was present at lower levels relative to Sk-BR-3 media control, while ERK2 and pan-AKT were comparable between both cell types. MEK1 and AKT2 appeared at slightly lower levels in K562 cells. GAPDH and vinculin levels were consistent across all samples, confirming equal loading and transfer quality. Together, these results confirm the specificity of each antibody, the absence of non-specific binding, and the suitability of both Sk-BR-3 and K562 cells as models for downstream proximity ligation assays aimed at monitoring dynamic signalling interactions.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Control, Positive Control, Western Blot, Molecular Weight, SDS Page, Fluorescence, Comparison, Derivative Assay, Expressing, Phospho-proteomics, Activation Assay, Binding Assay, Ligation

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: Analysis of primary blood cells from two patients diagnosed with CML, targeting molecular pathways known to be up-regulated in CML. The experiment used a slightly different oligonucleotide design compared to other experiments reported herein, but with similar performance ( .) A, E) Quantitative analysis of numbers of signals per cell revealed striking differences among individual blood cells. Top and bottom rows represent data for two different CML patients. Visualization cycle 1: phosphoPI3K-AKT1 in Cy3 (red), phosphoAKT1-AKT3 in Cy5 (green), AKT(pan)-AKT2 in FITC (yellow). Cycle 2: JAK2-JAK3 in Cy3 (blue), phosphorylated GRB2 in Cy5 (orange), MEK1-ERK in FITC (purple). Cycle 3: STAT3-phosphoSTAT3 in Cy5 (cyan), STAT3-phosphoSTAT3 in FITC (magenta) and STAT3-STAT5 in Cy3 (lime). The same color coding was used throughout all panels in the figure. B, F) Scatterplots of pairs of detection reactions, serving to visualize correlations across detection pairs (colors as in A, B)). C, G) Visualization of a zoomed-in view of detection reactions per cell. Outline of nuclei are shown in red. D, H) Cell to cell heterogeneity is visualized by overlaying individual detection reactions on the cells - here showing zoomed in region with D) phosphorylated GRB2 (in orange) and (h) STAT3-phosphoSTAT3 (in magenta). Cell nuclei are outlined in red. The raw image data from the full sample, together with detections are available for interactive viewing at https://ulflandegren2025.serve.scilifelab.se .

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: Interleukin-22 regulates gastric cancer cell proliferation through regulation of the JNK signaling pathway

doi: 10.3892/etm.2020.8707

Figure Lengend Snippet: Activation of the MAPK signaling pathway in AGS cells following transfection with IL-22 plasmid and IL-22 shRNA. (A) Representative western blots and (B) quantification results demonstrated that IL-22 plasmid decreased the phosphorylation of ERK and JNK, increased the phosphorylation of STAT3 and increased the expression of Bcl-2, whereas IL-22 shRNA increased the phosphorylation ERK and p-JNK, decreased the phosphorylation of STAT3 and decreased Bcl-2 expression. * P<0.05, ** P<0.01 and *** P<0.001 as indicated. MAPK, mitogen-activated protein kinase; IL, interleukin; shRNA, short hairpin RNA; p-, phosphorylated; t-, total.

Article Snippet: The anti-ERK monoclonal antibody (mab; MAB1230; 1:1,000), anti-phosphorylated (p)-ERK mab (MAB18251; 1:1,000), anti-JNK mab (AF1387; 1:2,000), anti-p-JNK mab (AF1205; 1:500), anti-STAT3 mab (MAB1799; 1:1,000), anti-p-STAT3 mab (AF4607; 1:500), anti-Bcl-2 mab (AF810; 1:1,000), anti-GAPDH mab (MAB5718; 1:2,000) and mouse or rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (HAF007, HAF008; 1:1,000) were purchased from R&D Systems, Inc.

Techniques: Activation Assay, Transfection, Plasmid Preparation, shRNA, Western Blot, Phospho-proteomics, Expressing