Digital gel documentation system

Immunoblotting was performed essentially as previously described (13 (link)). In brief, equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were blocked for 1 h with PBS containing 5% low fat milk powder (Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with primary antibodies anti-IL-1β 1:20,000 (kindly supplied by the National Cancer Institute, Frederick, MD), anti-Anx2 1:1,000 (Abnova, Taipai, Taiwan), anti-Arrb1 1:500 (Zymed, Carlsbad, CA), anti-Arrb2 1:2,000 (Abnova), anti-β-actin 1:100,000 (Sigma-Aldrich) or anti-GAPDH 1:10,000 (Novus Biologicals, Littleton, CO) in blocking solution. After extensive washing, the membranes were further incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, Glostrup, Denmark). For the detection of bound peroxidase, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used. Only β-actin and GAPDH were revealed with the less sensitive Lumi-Light substrate (Roche, Mannheim, Germany). To quantify the signal, densitometric analyses were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany).

Protein extracts of U937 cells were separated on 12% SDS-polyacrylamide gels under reducing conditions along with dual color precision plus protein standards (Bio-Rad, Hercules, CA, USA) and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% BSA diluted in PBS and incubated with polyclonal rabbit antibodies to P2Y1, P2Y11 (LS-C163318; 1:1000 and LS-C200442; 1:500; LifeSpan Biosciences via Biozol, Eching, Germany) or mouse monoclonal antibodies to β-actin (1:50000; A2228, Sigma-Aldrich). Primary antibodies were detected with horse radish peroxidase-labeled goat anti-rabbit Ig and rabbit anti-mouse Ig secondary antibodies (1:5000; Dako, Glostrup, Denmark), Lumi-Light substrate (Roche, Mannheim, Germany) and High Performance Chemiluminescence Films (GE Healthcare Bio-Sciences, Uppsala, Sweden). Documentation and densitometry were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany). Western blot analysis of IL-1β in concentrated cell culture supernatants was performed as described before [8 (link)].