antibody α-stat3  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Constitutive expression of the transcriptional co-activator IκBζ promotes melanoma growth and immunotherapy resistance

doi: 10.1038/s41467-025-60929-5

Figure Lengend Snippet: a – d In vitro cell proliferation was assessed using the CellTiter-Glo assay (Promega). For calculation, values obtained 24 h after initial seeding were set to 100%, and follow-up measurements were calculated as the percentage relative to the values from the first time point. IκBζ overexpression or knockdown, or phosphorylation of STAT3 (Y705) were analyzed by immunoblotting and normalized to β-Actin for each experiment. a Control or Nfkbiz knockout D4M-3A cells cultured in FCS-containing medium. b Control or IκBζ-overexpressing B16-F10 cells, cultured under starvation conditions (without FCS). c Control or IκBζ-overexpressing MV3 cells, cultured under starvation conditions (without FCS). d Same as in ( c ) with control cells cultured in the presence of supernatant from IκBζ-overexpressing MV3 cells that were previously cultured without FCS. Cell proliferation assay show the mean of 3 independent experiments ± standard deviation (SD). Significance was calculated using a two-tailed Student’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001, ns = not significant). Source data and exact P values are provided in the Source Data file.

Article Snippet: The following antibodies from Cell Signaling were used for immunoblot analysis: α-IκBζ (Cat. 9244), α-phospho-IκBα (Cat. 2859), α-IκBα (Cat. 9242), α-phospho-STAT1 (Y701, Cat. 9167), α-STAT1 (Cat. 14994), α-phospho-STAT3 (Y705, Cat. 9145), α-STAT3 (Cat. 4904), α-NF-κB1 p105/p50 (Cat. 3035), anti-NF-κB p65 (Cat. 8242), α-EZH2 (Cat. 5246), α-Tri-Methyl-Histone H3 (Lys27) (Cat. 9733), α-HDAC3 (Cat. 85057) and α-β-Actin (Cat. 3700).

Techniques: In Vitro, Glo Assay, Over Expression, Knockdown, Phospho-proteomics, Western Blot, Control, Knock-Out, Cell Culture, Proliferation Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Constitutive expression of the transcriptional co-activator IκBζ promotes melanoma growth and immunotherapy resistance

doi: 10.1038/s41467-025-60929-5

Figure Lengend Snippet: a Immunoblot analysis of total and phosphorylated STAT3 (Y705) and STAT1 (Y701), in control or IκBζ-depleted LOX-IMVI or YUMM1.7 cells, and in control or IκBζ-overexpressing B16-F10 cells. b IHC staining of phosphorylated STAT3 (Y705) in human melanoma samples. Top: Exemplified pictures of pSTAT3 staining in IκBζ-expressing and non-expressing tumors from patients showing either a complete response (CR) or progressive disease (PD) upon immunotherapy. Bottom: Correlation of the presence of IκBζ protein and pSTAT3 levels in tumor cells of tissue sections. c Chromatin fractionation of LOX-IMVI control or NFKBIZ knockdown cells. GAPDH and histone H3 served as internal controls for the chromatin-unbound and chromatin-bound fractions, respectively. d Chromatin immunoprecipitation of STAT3 and p65 in control or NFKBIZ knockdown LOX-IMVI cells. e Gene expression of IκBζ target genes in control or IκBζ-overexpressing MV3 cells, in the presence or absence of STAT3. STAT3 was lentivirally knocked down using shRNA, and control cells were generated using a non-coding shRNA (sh Ctrl). Gene expression was subsequently analyzed in the presence or absence of transient IκBζ overexpression and normalized to the reference gene RPL37A . Data derived from 3 independent experiments (mean ± standard deviation (SD)). Significance was calculated using a two-tailed Student’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001). Source data and exact P values are provided in the Source Data file.

Article Snippet: The following antibodies from Cell Signaling were used for immunoblot analysis: α-IκBζ (Cat. 9244), α-phospho-IκBα (Cat. 2859), α-IκBα (Cat. 9242), α-phospho-STAT1 (Y701, Cat. 9167), α-STAT1 (Cat. 14994), α-phospho-STAT3 (Y705, Cat. 9145), α-STAT3 (Cat. 4904), α-NF-κB1 p105/p50 (Cat. 3035), anti-NF-κB p65 (Cat. 8242), α-EZH2 (Cat. 5246), α-Tri-Methyl-Histone H3 (Lys27) (Cat. 9733), α-HDAC3 (Cat. 85057) and α-β-Actin (Cat. 3700).

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Expressing, Fractionation, Knockdown, Chromatin Immunoprecipitation, Gene Expression, shRNA, Generated, Over Expression, Derivative Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Constitutive expression of the transcriptional co-activator IκBζ promotes melanoma growth and immunotherapy resistance

doi: 10.1038/s41467-025-60929-5

Figure Lengend Snippet: a Immunoblot analysis of total and phosphorylated STAT3 (Y705) and STAT1 (Y701), in control or IκBζ-depleted LOX-IMVI or YUMM1.7 cells, and in control or IκBζ-overexpressing B16-F10 cells. b IHC staining of phosphorylated STAT3 (Y705) in human melanoma samples. Top: Exemplified pictures of pSTAT3 staining in IκBζ-expressing and non-expressing tumors from patients showing either a complete response (CR) or progressive disease (PD) upon immunotherapy. Bottom: Correlation of the presence of IκBζ protein and pSTAT3 levels in tumor cells of tissue sections. c Chromatin fractionation of LOX-IMVI control or NFKBIZ knockdown cells. GAPDH and histone H3 served as internal controls for the chromatin-unbound and chromatin-bound fractions, respectively. d Chromatin immunoprecipitation of STAT3 and p65 in control or NFKBIZ knockdown LOX-IMVI cells. e Gene expression of IκBζ target genes in control or IκBζ-overexpressing MV3 cells, in the presence or absence of STAT3. STAT3 was lentivirally knocked down using shRNA, and control cells were generated using a non-coding shRNA (sh Ctrl). Gene expression was subsequently analyzed in the presence or absence of transient IκBζ overexpression and normalized to the reference gene RPL37A . Data derived from 3 independent experiments (mean ± standard deviation (SD)). Significance was calculated using a two-tailed Student’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001). Source data and exact P values are provided in the Source Data file.

Article Snippet: The following antibodies from Cell Signaling were used for immunoblot analysis: α-IκBζ (Cat. 9244), α-phospho-IκBα (Cat. 2859), α-IκBα (Cat. 9242), α-phospho-STAT1 (Y701, Cat. 9167), α-STAT1 (Cat. 14994), α-phospho-STAT3 (Y705, Cat. 9145), α-STAT3 (Cat. 4904), α-NF-κB1 p105/p50 (Cat. 3035), anti-NF-κB p65 (Cat. 8242), α-EZH2 (Cat. 5246), α-Tri-Methyl-Histone H3 (Lys27) (Cat. 9733), α-HDAC3 (Cat. 85057) and α-β-Actin (Cat. 3700).

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Expressing, Fractionation, Knockdown, Chromatin Immunoprecipitation, Gene Expression, shRNA, Generated, Over Expression, Derivative Assay, Standard Deviation, Two Tailed Test

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: 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