Anti phosphatase cocktail

Microfluidic cultures were treated with 12-µL ice-cold lysis buffer: 5% deoxycholic acid (DOC, Sigma-Aldrich) in Tris-Buffered Saline (TBS), supplemented with Complete EDTA-free protease inhibitor (Roche) and anti-phosphatase cocktail (Sigma-Aldrich). Lysis buffer was directly injected in each microfluidic chamber and whole cell lysate collected. PAGE was performed with 4-12% NuPAGE polyacrylamide gels and MOPS buffer (Life Technologies). Proteins were blotted on PVDF membranes (Life Technologies) and detected with Carestream films (Kodak). A 1:1000 dilution of primary antibodies for Phospho-Akt (Ser 473), Akt, Phospho-AS160 (Thr 642), AS160 (all from Cell Signaling), IRS-1 (Millipore), GAPDH (Abcam), and HRP-conjugated secondary antibodies (mouse, Bio-Rad; rabbit, Life Technologies) were used. Gel images were quantified by standard image analysis, and ANOVA was performed to identify statistically significant differences.

Curcumin was purchased from Extrasynthese (France). Reagents for cell culture (RPMI 1640, penicillin/streptomycin, Trypsin/EDTA, Foetal Bovine Serum) were purchased from PAN biotech (Germany). Gelatin, paraformaldehyde (PFA), FITC-dextran, dimethylsulfoxide (DMSO), antiprotease cocktail, anti-phosphatase cocktail, Nonidet P40 and DAPI were obtained from Sigma-Aldrich (France). Recombinant-human-TNF- and MCP-1 were obtained from R&D systems and Miltenyi Biotec SAS (Paris, France) respectively.