Journal: bioRxiv

Article Title: FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-Cbl-NF-κB signaling

doi: 10.1101/2024.12.06.627217

Figure Lengend Snippet: HUVECs were starved for 12 h in 0.2% FBS DMEM with or without VEGF (5 ng/ml) prior to initiation of disturbed flow (5 dynes/cm 2 ). (A) Immunoblotting of active FAK (pY397), active NF-κB (pS536), active IKKα/β (pS176/177), IκBα, and β-actin as loading control are shown. (B) HUVECs were treated for 1 h with either DMSO or the FAK inhibitor (FAK-I, 2.5 μM) prior to initiation of disturbed flow (5 dynes/cm 2 ). Immunoblotting shows active FAK (pY397), active NF-κB (pS536), and β-actin as loading control. (C) Immunoblotting of CBL immunoprecipitates (IP) for CBL, FAK, and Tyrosine phosphorylation (4G10). (D) HUVECs were treated with FAK inhibitor (FAK-I, 2.5 μM) prior to initiation of disturbed flow (5 dynes/cm 2 ) for the indicated times. Immunostaining of pY397 FAK (Green) and CBL (Red). Nuclei were stained with DAPI (Blue). (E) HUVECs were treated with FAK inhibitor (FAK-I, 2.5 μM) prior to initiation of disturbed flow (5 dynes/cm 2 ) for the indicated times. Proximity ligation assay (PLA) was performed using antibodies for FAK and CBL. Co- localization is indicated by red dots. Nuclei were stained with DAPI (Blue).

Article Snippet: Anti-pY397 FAK (cat# 44-624G) was purchased from Life Technologies.

Techniques: Western Blot, Control, Immunostaining, Staining, Proximity Ligation Assay

Journal: bioRxiv

Article Title: FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-Cbl-NF-κB signaling

doi: 10.1101/2024.12.06.627217

Figure Lengend Snippet: Apoe-/- mice underwent partial carotid ligation (PCL) and were treated with either vehicle or FAK inhibitor (FAK-I, 35 mg/kg) while on a western diet (WD) for 2 weeks. The outer and inner aortic arches were immunostained for (A) active pY397 FAK (Red), (B) Cbl (Red), or (C) Vegfr2 (Red). Endothelial cells were stained with vWF (Green) and nuclei with DAPI (Blue).

Article Snippet: Anti-pY397 FAK (cat# 44-624G) was purchased from Life Technologies.

Techniques: Ligation, Western Blot, Staining

Journal: bioRxiv

Article Title: FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-Cbl-NF-κB signaling

doi: 10.1101/2024.12.06.627217

Figure Lengend Snippet: Partial carotid ligation (PCL) was performed on Apoe-/- mice and were treated with vehicle or FAK inhibitor (FAK-I, 30 mg/kg, twice daily) while on a western diet (WD) for 2 weeks. (A) Representative images of Oil Red O staining of sham and ligated carotid arteries are shown. Immunostaining of carotid arteries for (B) pY397 FAK (Red), (C) Cbl (Red), or (D) Vegfr2 (Red). Endothelial cells were stained with Vwf (Green) and nuclei with DAPI (Blue). (E and F) PLA was performed with antibodies targeting (E) FAK and Vegfr2 or (F) FAK and Cbl. Red dots indicate positive co-localization. Nuclei stained with DAPI (blue).

Article Snippet: Anti-pY397 FAK (cat# 44-624G) was purchased from Life Technologies.

Techniques: Ligation, Western Blot, Staining, Immunostaining

Journal: bioRxiv

Article Title: FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-Cbl-NF-κB signaling

doi: 10.1101/2024.12.06.627217

Figure Lengend Snippet: Partial carotid ligation (PCL) was performed on Apoe-/-;FAK-WT and Apoe-/-;FAK- KD mice and fed a western diet (WD) for 2 weeks. (A) Representative images of Oil Red O staining of sham and ligated carotid arteries are shown. Immunostaining for (B) pY397 FAK (Red), (C) Cbl (Red), or (D) Vegfr2 (Red). Endothelial cells stained with Vwf (Green) and nuclei were stained with DAPI (blue).

Article Snippet: Anti-pY397 FAK (cat# 44-624G) was purchased from Life Technologies.

Techniques: Ligation, Western Blot, Staining, Immunostaining

Journal: Cancer Gene Therapy

Article Title: Atypical cellular responses mediated by intracellular constitutive active TrkB (NTRK2) kinase domains and a solely intracellular NTRK2-fusion oncogene

doi: 10.1038/s41417-024-00809-0

Figure Lengend Snippet: Key resources table.

Article Snippet: Rabbit polyclonal anti- pY397-FAK (1:500) , Cell Signaling , Cat# 3283; RRID: AB_2173659.

Techniques: Western Blot, Recombinant, Staining, Membrane, Bicinchoninic Acid Protein Assay, Gel Extraction, Mutagenesis, Software

Journal: Virulence

Article Title: Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants

doi: 10.1080/21505594.2023.2249790

Figure Lengend Snippet: Anti-phagocytosis by Y. pseudotuberculosis is mediated through dephosphorylation of focal adhesion kinase. HeLa cells were infected with parental Y. pseudotuberculosis or strains producing the variants of YopD at a MOI of 20 ( a ) Representative confocal microscopy images of uninfected or infected HeLa cells with variants of Y. pseudotuberculosis . Arrowheads (white) shows FAK (pY397) stained focal adhesions (green). Nuclei were counterstained with DAPI (blue). Scale bars = 10 μm. (b) histogram indicates quantifications of focal adhesions per cell ( n = 25 cells), shown in ( a ). Representative data from two independent experiments is presented. Significance was determined using one-way analysis of variance (ANOVA) with Dunnett’s post-test against the parent strain of Y. pseudotuberculosis . **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Or ns = not significant. (c) at 10 min post infection, cells were lysed and FAK from each individual lysate was immunoprecipitated using anti-FAK antibodies bound to protein G-Sepharose. Equivalent amounts of eluted material was fractionated by SDS-PAGE, and following immunoblotting, membranes were incubated with anti-Phosphotyrosine antibody, clone 4G10, and then with HRP-conjugated anti-mouse antibodies. The displayed western blot image is one experiment. The scatter dot plot beneath this image represents the quantification achieved from four independent experiments ( n = 4), and represents the fold change in recovered phosphorylated FAK. The results presented are the mean value of four independent experiments. The quantification was carried out using ImageJ software. The triple asterisk (***), double asterisk (**) and single asterisk (*) reflects the degree of significant difference with p < 0.001, p < 0.01, and p < 0.05, respectively, when compared to the uninfected control. In an a parallel set of experiments, RAW 264.7 cells were infected with parental Y. pseudotuberculosis or strains producing the variants of YopD at a MOI of 20 ( d ). At 30 min post infection, the cells were fixed with 4% PFA. Extracellular bacteria were stained using Yersinia antisera followed by Alexa568-conjugated antibody. The cells were then permeabilized with 0.5% Triton X-100 and both extra- and intracellular bacteria were stained with Yersinia antisera followed by Alexa488-conjugated antibody. Cells were counterstained using Hoechst. For each sample, the amount of total and extracellular bacteria were counted manually using a fluorescence microscope over three independent experiments (i.e. 5 fields of view per independent experiment). The percent internalised bacteria was calculated for each field of view ( n = 15) and incorporated visualization of at least 150 eukaryotic cells per sample. The double asterisk (**) and single asterisk (*) reflect on the degree of significant difference with p < 0.01 and p < 0.0 5 , respectively, when compared to parental bacteria.

Article Snippet: The cells were incubated with primary antibodies, anti-phsopho (pY397)-FAK (BD Biosciences, #611723, 1:100 dilution), anti-phospho (Thr180/Tyr182)-p38 MAPK (Cell Signaling, #9211, 1:100 dilution) or anti-NF-κB (Santa Cruz, #sc -81,932, 1:100 dilution) in 10% FBS/PBS overnight at 4°C.

Techniques: De-Phosphorylation Assay, Infection, Confocal Microscopy, Staining, Immunoprecipitation, SDS Page, Western Blot, Incubation, Software, Bacteria, Fluorescence, Microscopy