Differential regulation of tyrosine phosphorylation on Eps8.

<p>Heavy, medium and light SILAC labelled HEK 293T cells were treated with either 25 nM dasatinib, 20 µM SU5402, or no inhibitor, prior to FGF2 stimulation (20 ng/ml; 15 min). Myc-Eps8 was immunoprecipitated and the resulting sample was run on an SDS-PAGE gel and, following in-gel trypsin digestion and phophopeptide enrichment, analysed by mass spectrometry. Each graph represents specific residues on Eps8 as indicated. Each data point represents a single peptide identification. <i>P</i> values were calculated by an unpaired t-test (0.01–0.05 =  *; 0.001–0.01 =  **; <0.001 =  ***). +, the median of the ratios isMethod S1). A) Experiment was carried out in the absence of sodium pervanadate. B) Experiment was carried out in the presence of 2 mM sodium pervanvadate.</p

A possible mechanism for the up-regulation of NADPH oxidase activity under redox stress.

<p>The pathway leading to the formation of GSH by the action of γ-glutamylcysteine synthetase (γGCS) is blocked by buthionine sulfoximine (BSO), inducing artificial stress condition in dHL60 cells. Decreased cellular GSH/GSSG ratio may activate redox sensitive enzymes such as ASMases and nuclear translocation of Nrf2. ASMases may favour lipid raft formation and thereby clustering active NADPH oxidase complexes in the outer membrane, subsequently liberating extracellular O₂^.-.</p

Effect of SFN on Nrf2 protein expression and activity in primary neutrophils.

<p>(A) Western blotting (left) and quantification (right) of total Nrf2 expression in the presence or absence of SFN treatment. (B) Western blotting (left) and quantification (right) of expression of Keap-1, Values are shown normalised to β-actin. (C) Analysis of Nrf2-DNA binding activity by trans-AM nuclear activity assay. A representative experiment of the three is shown. *** P< 0.01. (D) qPCR analysis of GCLC and GCLM gene expression levels of neutrophil mRNA from patients compared to control. (E) qPCR analysis of GCLC, HMOX1, GCLM and NQO1 gene expression levels in neutrophils treated with 5 µM SFN for 16 hours, values normalised to actin.</p

SFN pre-incubation increases the GSH/GSSG ratio in primary neutrophils.

<p>GSH (A) and GGSG (B) concentrations, along with the GSH/GSSG ratio (C) were measured using the DTNB recycling assay and expressed relative to protein content. Significant differences were calculated with Tukey’s multiple-comparison test where*P<0.05, and ***P<0.001.</p

SFN pre-incubation decreases neutrophil respiratory burst.

<p>Peak relative light unit (RLU) values for neutrophils stimulated with (A) 1µM fMLP, (B) opsonised <i>S. aureus</i> (300 bacteria/neutrophil) (C) <i>F. nucleatum</i> bacteria x100/neutrophil and (D) 10nM PMA in the presence or absence of SFN treatment were plotted and significant differences were calculated using Tukey’s multiple-comparison test, where, * p<0.05 and **p<0.01.</p

Chemical inhibition of Src kinase and FGFR kinase activity.

<p>A) HEK 293T cells were treated with SU5402, SU6656, or dasatinib 30 min prior to addition of FGF2 for 15 min. Cells were lysed and analysed by western blotting. B) HEK 293T cells were treated with increasing concentrations of dasatinib for 30 min prior to addition of FGF2 for 15 min. Cells were lysed and analysed by western blotting.</p

Eps8 and IRS4 interact in an FGF2 dependent manner that correlates with an increase in their tyrosine phosphorylation.

<p>A) HEK293T cells transfected with Myc-Eps8 were stimulated with 20 ng/ml FGF2 for 15 min either following 30 min pretreatment with SU5402 or dasatinib or in the absence of inhibitors. Anti-Myc immunoprecipitation and whole cell lysate samples were analysed by western blotting. B) HEK293T cells were stimulated with 20 ng/ml FGF2 for different lengths of time and tyrosine phosphorylated proteins were immunoprecipitated. Anti-pY immunoprecipitation and whole cell lysate samples were analysed by western blotting. C) HEK 293T cells were stimulated with 20 ng/ml FGF2 for 15 min and immnoprecipitations carried out using antibodies to either Eps8 or rabbit IgG. Resulting IP samples were analysed by western blotting. d) Following 30 min treatment with SU5402 or dasatinib and stimulation with 20 ng/ml FGF2 for a further 15 min, endogenous IRS4 was immunoprecipitated from HEK293T cells. Resulting IP samples were analysed by western blotting.</p

Effects of BSO and SFN pre-incubation on dHL60 cell GSH/GSSG ratio and respiratory burst.

<p>dHL60 cells were treated with 10µM BSO or 5µM SFN for 16 hours. GSH (A) and GSSG (B) concentrations were measured by the DTNB recycling assay in order to determine the GSH/GSSG ratio (C). Mean peak lucigenin chemiluminescence generated by dHL60 cells was plotted (RLU ± standard error of the mean) (D). Data represent three independent experiments of three replicates. Significant differences were calculated with one way ANOVA followed by Tukey’s multiple comparison test, where *P<0.05.</p

Effects of oxidative stress on ASMase activity and LR rearrangement.

<p>(A) BSO (10µM) pre-treated and untreated dHL60 cells were treated with or without desipramine (10 µM, 1 hour) and 5µM SFN. Cells (1×10⁷) were collected on ice-cold PBS, pelleted and lysed before analysing ASMase activity. (B) LRs from dHL60 cells were extracted with MNE buffer containing 1% triton X-100 and applied to discontinuous sucrose gradients, as described in Methods. Proteins were extracted and immunoblotted using anti-flotillin-1 as the raft protein detecting antibody. (C) Quantification of flotillin in lipid raft fractions 3 and 4 compared to total expression.</p

Protein-peptide interaction network for proteins binding specifically to phosphotyrosine-containing Eps8 peptides.

<p>A) Schematic diagram showing locations of the pY residues within the domain structure of Eps8. B) Using SILAC we carried out quantitative peptide pull-down assays from FGF2 stimulated (20 ng/ml; 15 min) HEK 293T cells to compare protein-peptide interactions for phosphotyrosine versus non-phosphotyrosine containing Eps8 peptides. Proteins interacting preferentially to phosphotyrosine peptides have been plotted in an interaction network.</p