Anti akt mab

Cellular extracts from MDMs treated with rNef were used to examine Akt and pAkt expression by Western blotting as described previously [39 (link)]. Anti-Akt-mAb (cat # 2967S, Cell Signaling Technologies, St Quentin, France), anti-pAkt (Thr308) mAb (cat # 2965S, Cell Signaling Technology), anti-pAkt (Ser473) mAb (cat # 4060S, Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) were used. Antiretroviral drugs (lopinavir/ritonavir and nevirapine) were obtained from the hospital pharmacy of the Centre Hospitalier Régional Universitaire (CHRU) Besançon. MDMs were treated for different periods of time with these drugs at indicated concentrations. The level of pAkt/Akt was determined by immunoblotting.

The murine LM8 and Dunn OS cell lines were kindly donated by Dr Eugenie Kleinerman (M.D. Anderson Cancer Center, Houston, TX, USA). The cell lines were cultured in high-glucose Dulbecco’s modified eagle’s medium (DMEM-h; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Recombinant murine CXCL12 was purchased from PeproTech (Rocky Hill, NJ, USA) and AMD3100 from Merck (Darmstadt, Germany). The rabbit antibodies (Ab) used for western blotting were obtained from different resources: anti-CXCR4 polyclonal Ab, anti-CXCR7 polyclonal Ab (both from Abcam, Cambridge, MA USA), anti-phospho-SAPK/JNK monoclonal Ab (MAb) (Thr183/Tyr185), anti-SAPK/JNK MAb, anti-phospho-c-Jun MAb (Ser73), anti-c-Jun MAb, anti-phospho-Akt MAb (Ser473), anti-Akt MAb, anti-phospho-p38 MAPK MAb (Thr180/Tyr182), anti-p38 MAPK MAb, anti-phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) MAb, anti-p44/42 MAPK (Erk1/2), anti-caspase-3 MAb, anti-caspase-8 MAb and anti-PARP MAb (all from Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, inc. Santa Cruz, CA, USA).