Journal: FEBS Open Bio

Article Title: Small molecule antagonism of oxysterol-induced Epstein–Barr virus induced gene 2 (EBI2) activation ☆

doi: 10.1016/j.fob.2013.02.003

Figure Lengend Snippet: (A) Chemical structure of GSK682753A. (B) Inhibition of [ 35 S]GTPγS binding to membranes from CHO cells stably expressing FLAG-tagged human EBI2 (solid squares) or pcDNA5 control (open circles) by GSK682753A in the presence of 1 nM 7α,25-OHC. The results are mean ± SEM of raw data from three independent experiments.

Article Snippet: 7α,25-OHC was purchased from Avanti Polar Lipids and GSK682753A was synthesized in-house at GlaxoSmithKline.

Techniques: Inhibition, Binding Assay, Stable Transfection, Expressing

Journal: FEBS Open Bio

Article Title: Small molecule antagonism of oxysterol-induced Epstein–Barr virus induced gene 2 (EBI2) activation ☆

doi: 10.1016/j.fob.2013.02.003

Figure Lengend Snippet: (A) 7α,25-OHC-induced [ 35 S]GTPγS binding to membranes from CHO cells stably expressing FLAG-tagged human EBI2 or pcDNA5 (open circles) at various concentrations of GSK682753A: no antagonist ([0], solid squares), 100 nM ([−7], solid diamonds), 1 μM ([−6], open squares) or 10 μM ([−5], open triangles). The results are mean ± SEM of raw data from three independent experiments. (B) Schild plot analysis of the dose–response curves presented in (A). The interception with the x -axis, which is an estimate of the log ( K d ) value is given. The slope of the linear regression is 1.4.

Article Snippet: 7α,25-OHC was purchased from Avanti Polar Lipids and GSK682753A was synthesized in-house at GlaxoSmithKline.

Techniques: Binding Assay, Stable Transfection, Expressing

Journal: FEBS Open Bio

Article Title: Small molecule antagonism of oxysterol-induced Epstein–Barr virus induced gene 2 (EBI2) activation ☆

doi: 10.1016/j.fob.2013.02.003

Figure Lengend Snippet: (A) 7α,25-OHC-induced β-arrestin2 recruitment in CHO cells transiently transfected with FLAG- and PK1-tagged human EBI2 wt (solid squares), F111Y mutant (open circles) or F111A mutant (open triangles). The results have been subtracted for background (pCMV transfected cells) and are given relative to the basal level, i.e. when no agonist is present, in percent as mean ± SEM. The data are from at least three independent experiments. (B) Inhibition of β-arrestin2 recruitment in CHO cells transiently transfected with FLAG- and PK1-tagged human EBI2 wt (solid squares), F111Y mutant (open circles) or F111A mutant (open triangles) by GSK682753A in the presence of 1 μM 7α,25-OHC. The results have been subtracted for background (pCMV transfected cells) and are given relative to the basal level, i.e. when 1 μM 7α,25-OHC but no antagonist is present, in percent as mean ± SEM. The data are from at least three independent experiments. (C) Ex vivo chemotaxis of B cells isolated from wt (open squares) or EBI2-overexpressing (solid squares) mice towards a gradient of 7α,25-OHC. The data are presented as chemotactic index as mean ± SEM and are from four independent experiments. (D) Inhibition of chemotaxis of EBI2-overexpressing B cells by GSK682753A in the presence of 0.1 nM 7α,25-OHC. The results are given relative to the basal level, i.e. when 0.1 nM 7α,25-OHC but no antagonist is present, in percent as mean ± SEM. The data are from three independent experiments.

Article Snippet: 7α,25-OHC was purchased from Avanti Polar Lipids and GSK682753A was synthesized in-house at GlaxoSmithKline.

Techniques: Transfection, Mutagenesis, Inhibition, Ex Vivo, Chemotaxis Assay, Isolation

Journal: FEBS Open Bio

Article Title: Small molecule antagonism of oxysterol-induced Epstein–Barr virus induced gene 2 (EBI2) activation ☆

doi: 10.1016/j.fob.2013.02.003

Figure Lengend Snippet: (A) 7α,25-OHC-induced ERK activation in serum-starved CHO cells stably expressing FLAG-tagged human EBI2. The data are presented as the ratio between phosphorylated ERK (pERK) and total ERK (tERK) as mean ± SEM from three independent experiments. Representative blots of pERK and tERK are shown below. Inset , ERK phosphorylation by 100 nM 7α,25-OHC in cells incubated overnight with 100 ng/mL pertussis toxin (+ptx) or DMSO vehicle (−ptx). (B) Inhibition of ERK activation by GSK682753A in the presence of 1 nM 7α,25-OHC. Presented as in (A). ( C ) 7α,25-OHC-induced p38 activation in serum-starved CHO cells stably expressing FLAG-tagged human EBI2 in after overnight incubation with pertussis toxin (+ptx, filled columns) or DMSO control (−ptx, empty columns). The data are presented as the ratio between phosphorylated p38 (p-p38) and total p38 (t-p38) as mean ± SEM from three independent experiments. Representative blots of p-p38 and t-p38 are shown below. * p lt 0.05 by Student's t -test.

Article Snippet: 7α,25-OHC was purchased from Avanti Polar Lipids and GSK682753A was synthesized in-house at GlaxoSmithKline.

Techniques: Activation Assay, Stable Transfection, Expressing, Incubation, Inhibition