egfr in vitro kinase assay  (BPS Bioscience)

Journal: Nature Communications

Article Title: EGFR-STAT1 pathway drives fibrosis initiation in fibroinflammatory skin diseases

doi: 10.1038/s41467-025-64648-9

Figure Lengend Snippet: A – C Wild type ( wt ) B6 ( n = 5) and Stat1 -/- mice ( n = 5) were injected with bleomycin (Bleo) subcutaneously and skin harvested on day 21 for histology ( A ), dermal skin thickness measurement ( B ) and collagen levels assayed by hydroxyproline content ( C ). D – F Stat1 -/- → wt ( n = 7) and wt → wt ( n = 8) bone marrow transplants were analyzed by FACS analysis of peripheral blood immune cells ( D ), then injected with bleomycin as in ( A ) and skin was analyzed for dermal thickness ( E ) and collagen ( F ). G , H Stat1 fl/fl / Pdgfra-Cre ( Stat1 ΔFib ) mice (PBS n = 5, Bleo n = 10) compared to Stat1 fl /fl controls (PBS n = 8, Bleo n = 6) were injected with bleomycin as in ( A ) and analyzed for dermal skin thickness ( G ) and histology ( H ). I, J Wt mice were injected with bleomycin subcutaneously and on the same day treated with the EGFR kinase inhibitor gefitinib (EGFRi) or JAK1/2 inhibitor ruxolitinib p.o. 5 days a week for 3 weeks (PBS n = 8, Bleo+Veh n = 12, Bleo+EGFRi n = 8, Bleo+JAK1/2i n = 7). Skin was analyzed for histology ( I ) and dermal thickness ( J ). Data in ( B , C , E , F , H , J ) are mean ± SD analyzed by two-way ANOVA ( B , C , E , F , H ) or one-way ANOVA ( J ) with Sidak’s multiple comparisons test ( B , C ), uncorrected Fisher’s LSD ( E , F , H ), or Tukey’s multiple comparisons test ( J ) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant). Source data are provided as a Source Data file.

Article Snippet: The EGFR in vitro kinase assay (BPS Bioscience 40321) was performed by incubating recombinant human STAT1 and recombinant human EGFR kinase in the kinase buffer (provided with the kit) supplemented with 100 μM ATP and 2 mM DTT.

Techniques: Injection

Journal: Nature Communications

Article Title: EGFR-STAT1 pathway drives fibrosis initiation in fibroinflammatory skin diseases

doi: 10.1038/s41467-025-64648-9

Figure Lengend Snippet: A – F Western blots of human foreskin fibroblasts (HFF) treated with EGFR ligands 100 ng/ml, IFNA2 1000 U/ml, or IFNG 1000 U/ml for 5 min unless otherwise noted. A STAT1, AKT and ERK phosphorylation in response to each EGFR ligand. B Time course of HFF treated with IFNA2 or TGFA. C HFF pretreated with or without gefitinib 5 μM for 30 min before addition of TGFA or EREG. D HFF treated with deucravacitinib (TYK2i) 0, 50 nM and 250 nM for 2 h before addition of IFNA2 or TGFA. E HFF treated with ruxolitinib (JAK1/2i) 0, 50 nM and 250 nM for 2 h before addition of IFNA2, IFNG or TGFA. F HFF treated with combined TYK2i and JAK1/2i before IFNA2 or TGFA. G In vitro kinase assay using recombinant EGFR kinase domain and recombinant STAT1 with increasing concentrations of gefitinib (EGFRi) analyzed for pSTAT1 (pY701) by western blot ( n = 3 per group). Quantification of the signal done by densitometry. H , I Proximity ligation assay (PLA) of HFF with and without TGFA 100 ng/ml for 5 min. Single antibody PLA was used as negative controls to account for any background signal from each antibody. PLA signal was quantified (I) as dots per cell in each 40x field across 5 replicates. Data in ( G , I ) are mean ± SD analyzed by one-way ANOVA with Tukey multiple-comparisons test (** p < 0.01, **** p < 0.0001, ns: not significant). All panels are representative of at least two independent experiments. GAPDH was used as the loading control in all western blots ( A – F ). Diagram of human icon created in BioRender. Odell, I. (2025) https://BioRender.com/emkz6wu . Source data are provided as a Source Data file.

Article Snippet: The EGFR in vitro kinase assay (BPS Bioscience 40321) was performed by incubating recombinant human STAT1 and recombinant human EGFR kinase in the kinase buffer (provided with the kit) supplemented with 100 μM ATP and 2 mM DTT.

Techniques: Western Blot, Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Proximity Ligation Assay, Control

Journal: Nature Communications

Article Title: EGFR-STAT1 pathway drives fibrosis initiation in fibroinflammatory skin diseases

doi: 10.1038/s41467-025-64648-9

Figure Lengend Snippet: A Time course of mNDF treated with TGFA 100 ng/ml compared to non-treated (NT) controls analyzed by qPCR, expression levels shown in heatmap. B Diagram of promoter regions and first three exons (blue boxes, not drawn to scale) of STAT1, overlapping EGR1/SP1, ELK4 and FOS binding sites. C–F Western blot ( C ) and qPCR (D – F , n = 4 per group ) of HFF pretreated with termuterkib (ERKi) or AZD8330 (MEKi) 100 nM prior to TGFA 100 ng/ml. G – K HFF pretreated with fludarabine (STATi) 100μM and analyzed by qPCR (G , I – K) and western blot ( H ) (G, n = 8 per group; I – K , n = 4 per group). L Diagram of the regulatory network between EGFR, ERK, EGR1 and STAT1 driving fibrotic gene expression. Mouse and human icons indicate from what organism the fibroblasts were isolated. Diagrams created in Biorender. Odell, I. (2025) https://BioRender.com/th7ial3 , https://BioRender.com/emkz6wu , https://BioRender.com/w2lhyzb . Data in ( D – F , I – K ) are mean ± SD analyzed by one-way ANOVA with Tukey multiple-comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant). Data in ( G ) is mean ± SD analyzed by two-tailed unpaired t -Test (** p < 0.01). Source data are provided as a Source Data file.

Article Snippet: The EGFR in vitro kinase assay (BPS Bioscience 40321) was performed by incubating recombinant human STAT1 and recombinant human EGFR kinase in the kinase buffer (provided with the kit) supplemented with 100 μM ATP and 2 mM DTT.

Techniques: Expressing, Binding Assay, Western Blot, Gene Expression, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: EGFR-STAT1 pathway drives fibrosis initiation in fibroinflammatory skin diseases

doi: 10.1038/s41467-025-64648-9

Figure Lengend Snippet: Activated keratinocytes defined by expression of S100A8 and stress keratins express high affinity EGFR ligands including TGFA. TGFA and possibly other high affinity ligands bind EGFR on fibroblasts, which phosphorylates STAT1 to induce a fibrotic expression profile. STAT1 acts as a master regulator of fibrosis genes independent of type I interferon signaling. Created in BioRender. Odell, I. (2025) https://BioRender.com/7b1sqwc .

Article Snippet: The EGFR in vitro kinase assay (BPS Bioscience 40321) was performed by incubating recombinant human STAT1 and recombinant human EGFR kinase in the kinase buffer (provided with the kit) supplemented with 100 μM ATP and 2 mM DTT.

Techniques: Expressing