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. 2016 Apr 14;6:24090. doi: 10.1038/srep24090

Figure 3. Nef triggers the activation of NF-ĸB and CD28RE via lipid raft mediated Akt signaling.

Figure 3

(A), Exogenous Nef activates Akt in TCR stimulated PBLs as measured by the detection of pAkt(Ser473). Five million PBLs were either left untreated or stimulated in various combinations: rNef only, anti-CD3-mAb only, anti-CD3-mAb + rNef (100 ng/ml), anti-CD28-mAb only, anti-CD28-mAb + rNef (100 ng/ml) and anti-CD3-mAb + anti-CD28-mAb) for 30 min. Expression of pAkt(Ser473 Thr308), total Akt and β-actin was determined by western blotting (n = 2). As control boiled rNef and rNef incubated with anti-Nef antibody were also included. (B,C), Detection of pAkt(Ser473) (B) and rNef (C) in lipid raft fractions isolated from PBLs treated with rNef. Five million PBLs were either left untreated or stimulated in various combinations: rNef only, anti-CD3-mAb only, anti-CD3-mAb + rNef (100 ng/ml), anti-CD28-mAb only, anti-CD28-mAb + rNef (100 ng/ml) and anti-CD3-mAb + anti-CD28-mAb) for 30 min. Cell lysates were prepared in 1% triton X-100 and subjected to ultracentrifugation over a sucrose gradient. Lipid rafts fractions were isolated and expression of pAkt (Ser 473), Nef and flotillin-1 were determined by western blotting (n = 5). Flotillin-1 was used as a lipid raft associated marker. (D), Akt-dependent NF-κB and CD28RE activation in PBLs treated with rNef. Five million PBLs were either left untreated or treated with rNef (100 ng/ml) for 30 min in the presence and absence of TCR stimulation (anti-CD3-mAb, anti-CD28-mAb), and pre-treated or not with the Akt inhibitor VIII for 2 hrs. Electrophoretic mobility shift assays was performed to measured CD28 responsive element and NF-kB activation in response to stimuli on a 6% native polyacrylamide gel as described in the materials and methods (n = 3).