Horseradish peroxidase conjugated anti rabbit or anti mouse igg

Total cellular protein was extracted by suspending the cells in SDS lysis buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol) together with freshly added proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Lysates were incubated on ice for 15 min. with intermittent mixing and then centrifuged at 5000 × g for 5 min. at 4°C. Supernatant was recovered and protein concentration was determined using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Western blot was performed by the enhanced chemiluminescence system. Briefly, extracted cellular proteins were separated by 10% SDS-polyacrylamide gels and electrotransferred onto polyvinylidine difluoride membranes. After blocking with 3% bovine albumin or 5% non-fat dry milk in PBS, the membranes were incubated with primary antibody. After washing, the membranes were probed with horseradish peroxidase-conjugated anti-rabbit or antimouse IgG, and the bands were visualized by the enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). The chemiluminescent signal is captured with a Fujifilm luminescent image LAS-1000 analyser (Fujifilm, Tokyo, Japan) and quantified with densitometric software Fujifilm Image Gauge. To confirm the equal protein loading, western blot for β-tubulin were performed. Quantitative measurements of the bands were done using ImageJ software.

Thrombin (Roche, Mannheim, Germany), convulxin (Axxora, Lörrach, Germany), Forskolin, Calyculin A, H89, thymoquinone, ODQ, IBMX, and SQ22536 (Sigma-Aldrich, Munich, Germany), DEA-NO (Alexis Biochemicals, Lörrach, Germany), Rp-8-Br-cAMPS and Rp-8-Br-PET-cGMPS (BioLog, Bremen, Germany), phospho-VASP^S239 (Nano Tools, Teningen, Germany), phospho Ser/Thr and actin antibodies (Cell Signaling, Frankfurt, Germany), horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham, Freiburg, Germany), Annexin-V-PE, P-selectin, rabbit anti-active caspase-3-PE, CD41-FITC, and PAC-1-FITC antibodies (BD-Bioscience, Heidelberg, Germany), JC1 (Invitrogen, Eugen, Germany), ABT-737 (Selleckchem, Munich, Germany), z-VAD-fmk and z-DEVD-fmk (Calbiochem, Schwalbach, Germany), calcein-AM (Molecular Probes, Gottingen, Germany) were purchased. PKA type II holoenzyme was purified from bovine heart as described.^{64 (link)} C-subunit of PKA antibody was a kind gift of G. Schwoch, Göttingen (Germany). Active caspase-3 (ENZO ALX-201-059) and caspase-3-specific substrate Ac-DEVD-pNA (ENZO ALX-260-033) were from ENZO Life Sciences (Lörrach, Germany).