rabbit α p stat3 y705  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Western blot analysis of CLIP1::ROS1-fusion expression and SHP2/MAPK and STAT3 signaling in CLIP1::ROS1-fusion iNHA; ΔTUB: abrogated microtubule interaction domain; GAPDH: loading control, p-ROS1 (Tyr2274) antibody used to validate fusion transgene activity, phospho-SHP2 (Tyr580), and p-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity and p-STAT3 (Tyr705), and p-STAT1 (Tyr701) for STAT activation. (B) Violin plots highlighting track mean speed from (C) for CCDC88A::ALK (left) and CLIP1::ROS1 (right); dots represent mean of biological replicates, significance calculated on mean values, significance calculated using unpaired two-tailed Student’s t-test, *: p-value ≤0.05. (C) Illustrative images of live cell tracking; inverted nuclear fluorescence, colored lines visualize tracks of individual cells within 12 hours; scalebar: 200µm. (D) Illustrative images of SIA assays; scalebar: 500µm. (E) SIA quantification of invading ALK- and ROS1-fusion iNHAs; left graph: number of invading cells, right graph: mean distance of invasion; one-way ANOVA (normally distributed) or Kruskal Wallis test (not normally distributed), post-hoc Dunn-Bonferroni test, *: p-value≤0.05, **: p-value <0.01,***:p-value<0.001, ****: p-value <0.0001. (F) SIA quantification of invading CCDC88A::ALK (first two graphs) or CLIP1::ROS1 (last two graphs) iNHAs treated with indicated STAT3i (Stattic) concentrations; first and third graph: number of invading cells, second and fourth graph: mean distance of invasion; one-way ANOVA (normally distributed) or Kruskal Wallis test (not normally distributed), post-hoc Dunn-Bonferroni test, *: p-value≤0.05, **: p-value <0.01, ****: p-value <0.0001.

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: Western Blot, Expressing, Control, Activity Assay, Activation Assay, Two Tailed Test, Cell Tracking Assay, Fluorescence

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Affinity purification MS/MS identifying direct interactors of ALK- and ROS1-fusions used in this study; size: −log 10 BFDR, color gradient: log 2 EFC high (red) to low (grey). (B) Immunoprecipitation validating SHC1/3 as direct interactors of ALK-fusion (top two blot) and SHP2 as direct interactor of ROS1-fusion (bottom three blots),respectively; GAPDH: loading control, p-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, p-SHC1 (Tyr239/240), and p-SHP2 (Tyr580) antibodies used to validate activity of interactors; dashed lines: marker lane. (C) Western blot analysis of MAPK signaling in CCDC88::ALK and CLIP1::ROS1 models. GAPDH: loading control, p-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, p-SHP2 (Tyr580), p-GAB1 (Tyr642), p-MEK1/2 (Ser217/221), and p-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity, p-STAT3 (Tyr705), and p-STAT1 (Tyr701) used to validate STAT activation. (D) Western blot analysis of RNAi effect in PPP1CB::ALK models. GAPDH: loading control, p-ALK (Tyr1507) antibody used to validate retained ALK activity, p-SHP2 (Tyr580) and p-GAB1 (Tyr642), used to validate shPTPN11 , p-STAT3 (Tyr705), used to validate shSTAT3 . (E) Kaplan-Meier survival curves showing tumor induced mortality upon orthotopic intracranial injection of shRNA inhibited PPP1CB::ALK cells in NSG mice, groups are represented by individual curves, with a n=8 mice per group,; grey: PPP1CB::ALK shCtrl , dark petrol: PPP1CB::ALK shSTAT3 , light petrol: PPP1CB::ALK shPTPN11 ; statistical significance determined by log-rank test, **: p-value<0.01, *:p-value<0.05.

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: Affinity Purification, Tandem Mass Spectroscopy, Immunoprecipitation, Control, Activity Assay, Marker, Western Blot, Activation Assay, Injection, shRNA

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) In vitro kinase assay validating SHP2 and STAT3 as substrates of ALK- and ROS1-fusions; GAPDH: loading control, phospho-ALK,-ROS1 antibody used to validate KD mutants, phospho-SHP2 (Tyr580) or phospho-STAT3 (Tyr705) validate ALK- and ROS1-fusion kinase specificity towards SHP2 or STAT3, respectively. (B) Western blot analysis of MAPK signaling in ALK- and ROS1-fusion models. GAPDH: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, phospho-SHP2 (Tyr580), phospho-GAB1 (Tyr642), phospho-MEK1/2 (Ser217/221), and phospho-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity, phospho-STAT3 (Tyr705), and phospho-STAT1 (Tyr701) used to validate STAT activation. (C) Subcellular fractionation of CLIP1::ROS1 samples validating increased STAT3 activity; phospho-ROS1 (Tyr2274) antibody used to validate KD mutant and phospho-STAT3 (Tyr705) used to validate pathway activity, β-TUB: cytoplasmic marker, H3: nuclear marker. (D) Western blots analyzing the effect of RTK inhibition (Entrectinib 500nM, 4hours) on MAPK and STAT signaling in ALK- and ROS1-fusion models. GAPDH: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate inhibition, phospho-SHP2 (Tyr580), phospho-GAB1 (Tyr642), phospho-MEK1/2 (Ser217/221), and phospho-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway inhibition and phospho-STAT3 (Tyr705), and phospho-STAT1 (Tyr701) for STAT inhibition.

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: In Vitro, Kinase Assay, Control, Western Blot, Activity Assay, Activation Assay, Fractionation, Mutagenesis, Marker, Inhibition

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Western blot analysis of ALK- and ROS1-fusion expression and Shp2/Mapk and Stat3 signaling in ALK- and ROS1-fusion IUE models. β-Actin: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate fusion transgene activity, phospho-Shp2 (Tyr580), phospho-Gab1 (Tyr642), phospho-Mek1/2 (Ser217/221), and phospho-Erk1/2 (Thr202/Tyr204) used to validate Mapk pathway activity and phospho-Stat3 (Tyr705) for Stat3 activation. (B) Western blots analyzing the effect of RTK inhibition (Entrectinib 100nM, 24hours) on Mapk and Stat3 signaling CLIP1::ROS1-fusion IUE models. β-Tub: loading control, phospho-ROS1 (Tyr2274) antibody used to validate inhibition, phospho-Shp2 (Tyr580), phospho-Gab1 (Tyr642), phospho-Mek1/2 (Ser217/221), and phospho-Erk1/2 (Thr202/Tyr204) used to validate Mapk pathway inhibition and phospho-Stat3 (Tyr705) for Stat3 inhibition. (C) Drug titration curves highlighting dose dependent effects of 72h treatment with Entrectinib (left), or Stattic (right) on IUE models, dark green: CCDC88A::ALK #235, light green: CCDC88A::ALK #236, berry: CLIP1::ROS1 #187; light berry: CLIP1::ROS1 #192, y-axis: linear drug concentrations, y-axis: survival normalized to DMSO control; dashed line: 50% survival; error bars: SD of 3 biological replicates. ( D) Unsupervised clustering, Euclidian distance with complete linkage heatmap of most variable transcripts, color gradient: z-score high (red) to low (blue), samples indicated at the top. (E) Enriched GO:terms for DEG in CCDC88A::ALK (upper) or GOPC::ROS1 (lower) samples. x-axis: −log 10 p-value significance established by ReViGo; left: GO:terms enriched in Entrectinib treated samples, right: GO:terms enriched in DMSO control samples

Article Snippet: IHC stainings with the following antibodies and according to manufacturer’s instructions were performed on an automated device (Leica, BOND; Roche, Ventana): mouse-α-ALK (1:10, 30min; Leica Biosystems #NCL-L-ALK), rabbit-α-p-ERK1/2 (1:200, 30min; CST #4370), rabbit-α-p-SHP2 (1:100, 30min; Invitrogen #PA5-114642), rabbit-α-p-STAT3 (1:100, 44min; CST #9145), rabbit-α-KI67 (1:40, 60min; Abcam #ab16667), and rabbit-α-ROS1 (1:100, 32min, Ventana; CST #63452).

Techniques: Western Blot, Expressing, Control, Activity Assay, Activation Assay, Inhibition, Titration

Journal: Frontiers in Cell and Developmental Biology

Article Title: Knockout of NOS2 Promotes Adipogenic Differentiation of Rat MSCs by Enhancing Activation of JAK/STAT3 Signaling

doi: 10.3389/fcell.2021.638518

Figure Lengend Snippet: Knockout of NOS2 promotes rat MSC adipocyte differentiation through signal transducer and activator of transcription (STAT)3 and JAK signaling. BMSCs from NOS2 –/– and WT rats at passage 3 were induced to differentiate into adipocytes and were collected to detect the expression of proteins in the JAK/STAT signaling pathway at the indicated time points. The protein levels of p-JAK2, JAK2, p-STAT1, –3, and –5 and STAT1, –3, and –5 in NOS2 –/– and WT rat BMSCs were assessed by western blotting. (A) Representative western blots are shown. (B–E) Density ratios of p-JAK2 and JAK2, p-STAT1 and STAT1, p-STAT3 and STAT3, and p-STAT5 and STAT5. Data are normalized to GAPDH. All results are expressed as the means ± SEM; * P < 0.05; ** P < 0.01; n = 4.

Article Snippet: The membranes were blocked in 5% non-fat milk (in Tris-buffered saline containing 0.1% Tween-20) for 1.5 h and then incubated with primary antibodies [α-phosphorylated (p)-STAT1 (1:1,000, #7649), α-p-STAT3 (1:1,000, #9145), α-p-STAT 5 (1:1,000, #4322), α-STAT1 (1:1,000, #14994), α-STAT3 (1:1,000, #9139), α-STAT 5 (1:1,000, #94205), α-GAPDH (1:1,000, #5174), α-p-JAK2 (1:1,000, #3776), α-JAK2 (1:1,000, #3230) from Cell Signaling Technology (Danvers, MA, United States) and α-PPAR-γ (1:500, #ab209350), α-NOS2 (1:500, #ab3523) from Abcam (Cambridge, MA, United States)] and then with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Knock-Out, Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Knockout of NOS2 Promotes Adipogenic Differentiation of Rat MSCs by Enhancing Activation of JAK/STAT3 Signaling

doi: 10.3389/fcell.2021.638518

Figure Lengend Snippet: AG490 and S3I-201 inhibit STAT3 activation and adipocyte differentiation in NOS2 –/– rat MSCs. BMSCs from NOS2 –/– rats at passage 3 were induced to differentiate into adipocytes in the presence or absence of AG490 (10 μM) and S3I-201 (20 μM) and were collected to detect p-STAT3 and STAT3 expression levels (A,B) and adipocyte differentiation (C,D) on d 12 after differentiation. (E) Representative western blots are shown in WAT from WT and NOS2 –/– SD rats after HFD feeding. (F) Density ratios of p-STAT3 and STAT3. The scale bar indicates 100 μm. All results are expressed as the means ± SEM; ** P < 0.01; n = 4.

Article Snippet: The membranes were blocked in 5% non-fat milk (in Tris-buffered saline containing 0.1% Tween-20) for 1.5 h and then incubated with primary antibodies [α-phosphorylated (p)-STAT1 (1:1,000, #7649), α-p-STAT3 (1:1,000, #9145), α-p-STAT 5 (1:1,000, #4322), α-STAT1 (1:1,000, #14994), α-STAT3 (1:1,000, #9139), α-STAT 5 (1:1,000, #94205), α-GAPDH (1:1,000, #5174), α-p-JAK2 (1:1,000, #3776), α-JAK2 (1:1,000, #3230) from Cell Signaling Technology (Danvers, MA, United States) and α-PPAR-γ (1:500, #ab209350), α-NOS2 (1:500, #ab3523) from Abcam (Cambridge, MA, United States)] and then with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: IKKβ acts as a tumor suppressor in cancer-associated fibroblasts during intestinal tumorigenesis

doi: 10.1084/jem.20150576

Figure Lengend Snippet: Fibroblast-specific Ikkβ deletion promotes colon tumorigenesis. (A) Tumor incidence in Ikkβ F/F and Ikkβ ΔFib mice at the end of the AOM/DSS regimen on day 84. Data are mean from two independent experiments ± SE; n ≥ 7; ns, not significant. (B) Mean tumor size in Ikkβ F/F and Ikkβ ΔFib mice. Data are mean ± SE; n ≥ 7; ***, P < 0.0001 by Student’s t test. (C) Histogram showing size distribution of tumors in Ikkβ F/F and Ikkβ ΔFib mice. (D and E) Overview of representative sections of the Swiss rolls that were used for tumor counting; bars, 1 mm. (F–H) Immunohistochemical analysis of BrdU incorporation (F and G) and proliferation index (H) in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bars, 50 µm. (I) Quantification of cleaved caspase 3 staining for the apoptotic index in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. (J) Tumor incidence in Ikkβ F/F and Ikkβ ΔFib mice at the end of the sporadic tumor regimen (six weekly AOM injections without additional DSS administration) at week 22. Data are mean from two independent experiments ± SE; n ≥ 9; ns, not significant. (K) Mean tumor size in Ikkβ F/F and Ikkβ ΔFib mice. Data are mean ± SE; n ≥ 9; **, P < 0.005 by Student’s t test. (L) Quantification of the proliferation index in Ikkβ F/F and Ikkβ ΔFib tumors. Data are mean ± SE; n ≥ 20 tumors per genotype; *, P < 0.05 by Student’s t test. (M and N) Quantification of blood vessel FCD length and area in tumors from fluorescein injected Ikkβ F/F and Ikkβ ΔFib mice by CLSM. Data are mean ± SE; n ≥ 6. ***, P < 0.0001 by Student’s t test. (O–Q) Quantitative analysis of EpCAM − CD45 − CD31 + endothelial cells (O), EpCAM − CD45 − PDGFRα + fibroblasts (P), and CD45 + leukocytes (Q) in tumors from Ikkβ F/F and Ikkβ ΔFib mice analyzed by flow cytometry. Data are mean ± SE; n ≥ 6. *, P < 0.05 by Student’s t test. (R–T) Immunohistochemical analysis of p-Stat3 Y705 (R and S) and quantification (T) in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bar, 200 µm. (U–W) Immunohistochemical analyses of p-Akt S473 (U and V) and quantification (W) in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bar, 300 µm. (X–Z) Immunohistochemical analysis of β-catenin (X and Y) and quantification (Z) of nuclear expression in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bars, 300 µm. Data in H, I, T, W, and Z are mean ± SE; n ≥ 10 tumors of each genotype; *, P < 0.05; ***, P < 0.0001 by Student’s t test.

Article Snippet: The following primary antibodies were used: α-BrdU (MCA2060; AbDSerotec), α-cleaved caspase 3 (9661S; Cell Signaling Technology), α-p-Stat3 Y705 (9145; Cell Signaling Technology), α-pAkt S473 (3787S; Cell Signaling Technology), α-β-catenin (06–734; EMD Millipore), α-p-Met (ab5662; Abcam), α-E-cadherin (610182; BD).

Techniques: Immunohistochemical staining, BrdU Incorporation Assay, Staining, Injection, Flow Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: IKKβ acts as a tumor suppressor in cancer-associated fibroblasts during intestinal tumorigenesis

doi: 10.1084/jem.20150576

Figure Lengend Snippet: Met inhibition prevents tumor promotion in Ikkβ ΔFib mice upon CAC challenge. (A) Schematic overview of the CAC model and mode of capmatinib application. During DSS administration, capmatinib was paused and mice received tamoxifen containing AIN-76A diet. (B and C) Tumor incidence (B) and average tumor size (C) in Ikkβ F/F and Ikkβ ΔFib mice at the end of the AOM/DSS regimen that received capmatinib or were left untreated. Data are mean ± SE; n ≥ 7; **, P < 0.005; ***, P < 0.0001 by ANOVA, followed by Bonferroni post hoc test for multiple datasets. (D) Quantification of blood vessel FCD length and area in tumors from capmatinib-treated Ikkβ F/F and Ikkβ ΔFib mice by CLSM. Data are mean ± SE; n ≥ 6; ns, not significant. (E) Quantification of immunohistochemical analysis of p-Akt S473 in tumor epithelial cells from untreated and capmatinib treated Ikkβ ΔFib mice. (F) Quantification of immunohistochemical analysis of p-Stat3 Y705 in tumor epithelial cells from untreated and capmatinib-treated Ikkβ ΔFib mice. (G) Quantification of immunohistochemical analysis of nuclear β-catenin in tumor epithelial cells from untreated and capmatinib-treated Ikkβ ΔFib mice. Data in E–G are mean ± SE; n ≥ 10 tumors of each genotype; *, P < 0.05; ***, P < 0.0001 by Student’s t test. (H) Flow cytometric analysis of lamina propia lymphocytes in Ikkβ F/F and Ikkβ ΔFib mice on day 15 of the CAC model revealed no difference in the numbers recruited CD3 + CD4 + , CD4 + FoxP3 + , and CD4 + IFNγ + when the mice received a diet containing the Met inhibitor capmatinib together with tamoxifen. Data are mean ± SE; n ≥ 7 mice per group; ns, not significant.

Article Snippet: The following primary antibodies were used: α-BrdU (MCA2060; AbDSerotec), α-cleaved caspase 3 (9661S; Cell Signaling Technology), α-p-Stat3 Y705 (9145; Cell Signaling Technology), α-pAkt S473 (3787S; Cell Signaling Technology), α-β-catenin (06–734; EMD Millipore), α-p-Met (ab5662; Abcam), α-E-cadherin (610182; BD).

Techniques: Inhibition, Immunohistochemical staining