The lipid-modulating effects of a CD4-specific recombinant antibody correlate with ZAP-70 segregation outside membrane rafts

Abstract

To cite this version:Myriam Chentouf, Maxime Rigo, Soufiane Ghannam, Isabelle Navarro-Teulon, Sébastien Mongrand, et al.. The lipid-modulating effects of a CD4-specific recombinant antibody correlate with ZAP-70 segregation outside membrane rafts..

CD4 ligation excludes the Carma1–Bcl10–MALT1 complex from GM1-positive membrane rafts in CD3/CD28 activated T cells

“…After washing in PBS-T, binding was revealed with the ECL Western Blotting Detection Kit (Amersham) [4]. Neither the experiments performed for this paper, nor the dot inserm-00588709, version 1 -26 Apr 2011 blots/western blots we previously published [4,5] showed GM1 and CD71 reactivity in fractions 1-3 of the membrane raft gradient.…”

“…After washing in 160 mM PBS (pH 7.4), cells were lysed at 37°C in 1% Brij 98 detergent diluted in TNE buffer (25 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA and 1 mg/ml enzyme inhibitors) (complete EDTA-free mixture of anti-proteases; Roche) for 30 min. Cell lysates were mixed with an equal volume of 80% sucrose in TNE, overlaid with 6.5 ml of 30% and 3.5 ml of 5% sucrose in TNE, and then centrifuged at 200,000 g for 20 h [4,5]. Twelve 1-ml fractions were collected on ice starting from the top of the gradient and numbered from 1 to 12.…”

“…Twelve 1-ml fractions were collected on ice starting from the top of the gradient and numbered from 1 to 12. The protein content of each fraction was quantified with the micro Bradford Protein Assay kit (Pierce) [4,5]. GM1 and CD71 expression were used to discriminate between rafts and non-rafts fractions (see below).…”

“…Specifically, the rIgG 1 13B8.2 antibody induces CD4 accumulation in GM1 † -positive membrane rafts and reorganization of signalling proteins involved in TCR-mediated immune response [4], through recruitment of CD3, Lyn, p56 Lck and LAT and exclusion of ZAP-70, SLP-76, PLC1 and Vav-1 from membrane rafts of Jurkat T cells [4]. Moreover, 13B8.2 increases ceramide release through acid sphingomyelinase activation and decreases phosphatidylserine synthesis without modifying the cholesterol content of GM1-positive membrane rafts [5].…”

Pathways of polyunsaturated fatty acid utilization: Implications for brain function in neuropsychiatric health and disease

“…N-3 PUFAs also consistently have been implicated in lipid raft regulation of immunomodulation (Ariel et al, 2005; Basiouni et al, 2012; Baumer et al, 2010; Bonilla et al, 2010; Chentouf et al, 2010; De Smedt-Peyrusse et al, 2008; Diaz et al, 2002; Geyeregger et al, 2005; Gurzell et al, 2013; Hou et al, 2012; Kim et al, 2008b; Li et al, 2005; Li et al, 2007; Li et al, 2008; Murakami et al, 2012; Ruth et al, 2009; Schumann et al, 2011; Teague et al, 2013; Van Laethem and Leo, 2002; Webb et al, 2000; Wong et al, 2009a; Zeyda et al, 2002), including a direct influence on lipid raft modulation of Toll-like receptor 4, a factor governing induction of genes that regulate inflammatory responsiveness (Wong et al, 2009a). …”