Anti phosho iκbα

The following antibodies were used for flow cytometry analysis: anti-Apo2.7-PE, anti-CD5-FITC, anti-CD19-APC and control IgG1-FITC mAbs were purchased from BD Biosciences (Le Pont de Claix, France). Analysis of protein expression was conducted by immunoblotting using the following primary antibodies: anti-Bcl-2, anti-IκBα and anti-phosho-IκBα (Cell Signaling, Saint Quentin en Yvelines, France), Anti-Mcl-1 (S19) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl-x_L (BD Biosciences, Le Pont de Claix, France), Anti-NF-κB p52 Antibody and anti-actin (Merck Millipore, Lyon, France). ABT-199 was kindly provided by Abbvie Laboratories (North Chicago, IL, USA) and the selective BTK inhibitor ibrutinib (PCI-32765) was obtained from Selleck Chemicals (Souffelweyersheim, France).

We isolated bone marrow cells and cultured them with 10% L-929 cell culture medium as described previously [15 (link)]. We plated BMMϕs in 35 mm dishes and cultured them for 12 hours. We then added live or heat inactivated V. vulnificus at the indicated multiplicity of infection (MOI) or V. vulnificus lysates to the cells. We collected supernatants at the indicated times for cytokine quantification. We then subjected the infected cells to lysate preparation and Western blot analysis by following a previous protocol [41 (link)]. Anti-phosho-mTOR, anti-phosho-S6K1, anti-phosho-S6, anti-phosho-Akt, anti-phosho-4EBP1, anti-phosho-IKKα/β, anti-phosho-IκBα, and anti-β actin antibodies all came from Cell Signaling Technology.