mouse α stat3  (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: Primary antibodies used: rabbit-α-ALK (1:60, CST #3633), rabbit-α-ROS1 (1:80, CST #63452), rabbit-α-SHP2 (1:40, CST #3397), rabbit-α-p-SHP2(Y580) (1:60, Thermo Fisher #PA5-114642), rabbit-α-ERK1/2 (1:150, CST #4695), rabbit-α-p-ERK1/2(T202, Y204) (1:80, CST #4370), mouse-α-STAT3 (1:40, CST #9139), rabbit-α-p-STAT3(Y705) (1:60, CST #9145), rabbit-α-ACTIN (1:300, Abcam #ab8227), mouse-α-LaminB1 (1:80, Biolegend #869802), rat-α-KI67 (1:200, Thermo Fisher #17-5698-82), rabbit-α-H3K27me3 (1:40, Abcam #ab192985), rabbit-α-H3K27ac (1:40, Abcam #ab4729), rabbit-α-p-AKT(S437) (1:80, CST #4060), rat-α-SOX2 (1:80, Thermo Fisher # 14-9811-82), mouse-α-NESTIN (1:20, BD-Biosciences #556309), goat-α-SOX10 (1:80, R&D #AF2864), chicken-α-GFAP (1:1500, NovusBio #NBP1-05198), goat-α-OLIG2 (1:20, R&D #AF2418), goat-α-PDGFRα (1:200, R&D #AF1062), rabbit-α-S100β (1:1300, Abcam #ab52642), and rat-α-MBP (1:1500, Abcam #ab7349).

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: Primary antibodies used: rabbit-α-ALK (1:60, CST #3633), rabbit-α-ROS1 (1:80, CST #63452), rabbit-α-SHP2 (1:40, CST #3397), rabbit-α-p-SHP2(Y580) (1:60, Thermo Fisher #PA5-114642), rabbit-α-ERK1/2 (1:150, CST #4695), rabbit-α-p-ERK1/2(T202, Y204) (1:80, CST #4370), mouse-α-STAT3 (1:40, CST #9139), rabbit-α-p-STAT3(Y705) (1:60, CST #9145), rabbit-α-ACTIN (1:300, Abcam #ab8227), mouse-α-LaminB1 (1:80, Biolegend #869802), rat-α-KI67 (1:200, Thermo Fisher #17-5698-82), rabbit-α-H3K27me3 (1:40, Abcam #ab192985), rabbit-α-H3K27ac (1:40, Abcam #ab4729), rabbit-α-p-AKT(S437) (1:80, CST #4060), rat-α-SOX2 (1:80, Thermo Fisher # 14-9811-82), mouse-α-NESTIN (1:20, BD-Biosciences #556309), goat-α-SOX10 (1:80, R&D #AF2864), chicken-α-GFAP (1:1500, NovusBio #NBP1-05198), goat-α-OLIG2 (1:20, R&D #AF2418), goat-α-PDGFRα (1:200, R&D #AF1062), rabbit-α-S100β (1:1300, Abcam #ab52642), and rat-α-MBP (1:1500, Abcam #ab7349).

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: Primary antibodies used: rabbit-α-ALK (1:60, CST #3633), rabbit-α-ROS1 (1:80, CST #63452), rabbit-α-SHP2 (1:40, CST #3397), rabbit-α-p-SHP2(Y580) (1:60, Thermo Fisher #PA5-114642), rabbit-α-ERK1/2 (1:150, CST #4695), rabbit-α-p-ERK1/2(T202, Y204) (1:80, CST #4370), mouse-α-STAT3 (1:40, CST #9139), rabbit-α-p-STAT3(Y705) (1:60, CST #9145), rabbit-α-ACTIN (1:300, Abcam #ab8227), mouse-α-LaminB1 (1:80, Biolegend #869802), rat-α-KI67 (1:200, Thermo Fisher #17-5698-82), rabbit-α-H3K27me3 (1:40, Abcam #ab192985), rabbit-α-H3K27ac (1:40, Abcam #ab4729), rabbit-α-p-AKT(S437) (1:80, CST #4060), rat-α-SOX2 (1:80, Thermo Fisher # 14-9811-82), mouse-α-NESTIN (1:20, BD-Biosciences #556309), goat-α-SOX10 (1:80, R&D #AF2864), chicken-α-GFAP (1:1500, NovusBio #NBP1-05198), goat-α-OLIG2 (1:20, R&D #AF2418), goat-α-PDGFRα (1:200, R&D #AF1062), rabbit-α-S100β (1:1300, Abcam #ab52642), and rat-α-MBP (1:1500, Abcam #ab7349).

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: Primary antibodies used: rabbit-α-ALK (1:60, CST #3633), rabbit-α-ROS1 (1:80, CST #63452), rabbit-α-SHP2 (1:40, CST #3397), rabbit-α-p-SHP2(Y580) (1:60, Thermo Fisher #PA5-114642), rabbit-α-ERK1/2 (1:150, CST #4695), rabbit-α-p-ERK1/2(T202, Y204) (1:80, CST #4370), mouse-α-STAT3 (1:40, CST #9139), rabbit-α-p-STAT3(Y705) (1:60, CST #9145), rabbit-α-ACTIN (1:300, Abcam #ab8227), mouse-α-LaminB1 (1:80, Biolegend #869802), rat-α-KI67 (1:200, Thermo Fisher #17-5698-82), rabbit-α-H3K27me3 (1:40, Abcam #ab192985), rabbit-α-H3K27ac (1:40, Abcam #ab4729), rabbit-α-p-AKT(S437) (1:80, CST #4060), rat-α-SOX2 (1:80, Thermo Fisher # 14-9811-82), mouse-α-NESTIN (1:20, BD-Biosciences #556309), goat-α-SOX10 (1:80, R&D #AF2864), chicken-α-GFAP (1:1500, NovusBio #NBP1-05198), goat-α-OLIG2 (1:20, R&D #AF2418), goat-α-PDGFRα (1:200, R&D #AF1062), rabbit-α-S100β (1:1300, Abcam #ab52642), and rat-α-MBP (1:1500, Abcam #ab7349).

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

Journal: iScience

Article Title: Cannabinoid receptor type 1 (CB 1 R) inhibits hypothalamic leptin signaling via β-arrestin1 in complex with TC-PTP and STAT3

doi: 10.1016/j.isci.2023.107207

Figure Lengend Snippet: Feeding-induced hypothalamic signaling by endogenous leptin is impeded by CB 1 R activation in a β-arrestin1-dependent manner (A) Scheme of the fasting-refeeding paradigm. After an overnight fast, food was made available ad libitum for 2 or 4 h. Then, food was again removed and mice were treated with the CB 1 R agonist ACEA (2 or 10 μM, i.p. ) or with vehicle for 45 or 90 min and then euthanized under anesthesia. (B) Immunohistochemical analysis of phospho-STAT3 in the arcuate nucleus (approx. bregma −2 mm). n = 64–594-586 phospho-STAT3 + nuclei (L to R) from 4-6-6 animals (respectively); ∗p < 0.0001 vs. non-refed group and p#<0.0001 vs. refeeding + /ACEA − (ANOVA followed by Kruskal-Wallis post-hoc test). (C) Left bar graph : serum leptin concentration in WT and CB 1 R knockout animals subjected to the fasting-refeeding paradigm; n = 6-6-5-9-6-7 (L to R); ∗p < 0.05 and ∗∗p < 0.01 compared to refeeding − /ACEA − group of the same genetic background (ANOVA with Kruskal-Wallis post-hoc test). Middle panel : representative western blot showing hypothalamic STAT3 activation in WT and CB 1 R −/− animals. Right panel : statistical analysis of pooled western blot data; n = 11–14-12-7-5-4 (L to R); ∗∗p < 0.001 and ∗p < 0.01 vs. WT non-refed group, # p < 0.01 vs. refeeding + /ACEA − , $$ p < 0.01 and $ p < 0.05 compared to CB 1 R −/− non-refed group (ANOVA and Holm-Sidak’s or Dunn’s post-hoc tests). (D) Hypothalamic β-arrestin1 expression (left panel) and STAT3 activation (middle and right panels) in fasting-refeeding experiments in β-arrestin1 knockout mice. Number of observations was 5 in all groups; ∗p < 0.039 vs. non-refed group (ANOVA and Sidak’s post-hoc test). Mean + S.E.M. are shown.

Article Snippet: α-pSTAT3 (serine), monoclonal , Cell Signaling Technology , Cat#9136; RRID: AB_331755.

Techniques: Activation Assay, Immunohistochemical staining, Concentration Assay, Knock-Out, Western Blot, Expressing

Journal: iScience

Article Title: Cannabinoid receptor type 1 (CB 1 R) inhibits hypothalamic leptin signaling via β-arrestin1 in complex with TC-PTP and STAT3

doi: 10.1016/j.isci.2023.107207

Figure Lengend Snippet:

Article Snippet: α-pSTAT3 (serine), monoclonal , Cell Signaling Technology , Cat#9136; RRID: AB_331755.

Techniques: Recombinant, Diagnostic Assay, Enzyme-linked Immunosorbent Assay, Produced, Control, Software, Translocation Assay, Imaging, Western Blot