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Image Search Results
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: Antibodies used in this study: mouse-α-ALK (CST #3791), rabbit-α-p-ALK(Y1507) (CST #14678), mouse-α-ROS1 (CST #3266), rabbit-α-p-ROS1(Y2274) (CST #3078), mouse-α-SHP2 (Abcam #ab76285), rabbit-α-p-SHP2(Y580) (CST #5431), mouse-α-MEK1/2 (CST #4694), rabbit-α-p-MEK1/2 (S217/221) (CST #9154), mouse-α-ERK1/2 (CST #4696), rabbit-α-p-ERK1/2(T202,Y204), rabbit-α-GAB1 (CST #3232), rabbit-α-p-GAB1(Y627) (CST #3233), mouse-α-STAT3 (CST #9139), rabbit-α-p-STAT3(Y705) (CST #9145), rabbit-α-p-STAT3(Y705) ( in vitro kinase only; Abcam #ab267373),
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: Antibodies used in this study: mouse-α-ALK (CST #3791), rabbit-α-p-ALK(Y1507) (CST #14678), mouse-α-ROS1 (CST #3266), rabbit-α-p-ROS1(Y2274) (CST #3078), mouse-α-SHP2 (Abcam #ab76285), rabbit-α-p-SHP2(Y580) (CST #5431), mouse-α-MEK1/2 (CST #4694), rabbit-α-p-MEK1/2 (S217/221) (CST #9154), mouse-α-ERK1/2 (CST #4696), rabbit-α-p-ERK1/2(T202,Y204), rabbit-α-GAB1 (CST #3232), rabbit-α-p-GAB1(Y627) (CST #3233), mouse-α-STAT3 (CST #9139), rabbit-α-p-STAT3(Y705) (CST #9145), rabbit-α-p-STAT3(Y705) ( in vitro kinase only; Abcam #ab267373),
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: Antibodies used in this study: mouse-α-ALK (CST #3791), rabbit-α-p-ALK(Y1507) (CST #14678), mouse-α-ROS1 (CST #3266), rabbit-α-p-ROS1(Y2274) (CST #3078), mouse-α-SHP2 (Abcam #ab76285), rabbit-α-p-SHP2(Y580) (CST #5431), mouse-α-MEK1/2 (CST #4694), rabbit-α-p-MEK1/2 (S217/221) (CST #9154), mouse-α-ERK1/2 (CST #4696), rabbit-α-p-ERK1/2(T202,Y204), rabbit-α-GAB1 (CST #3232), rabbit-α-p-GAB1(Y627) (CST #3233), mouse-α-STAT3 (CST #9139), rabbit-α-p-STAT3(Y705) (CST #9145), rabbit-α-p-STAT3(Y705) ( in vitro kinase only; Abcam #ab267373),
Techniques: In Vitro, Kinase Assay, Control, Western Blot, Activity Assay, Activation Assay, Fractionation, Mutagenesis, Marker, Inhibition