Figure 7
No impact of ABT-737 and TQ on cyclic nucleotide levels or phosphatase activity. (a,b) Washed human platelets (3 × 108/ml) were incubated with indicated concentrations of ABT-737 for 60 min (a), or for the indicated time with 40 μM TQ (b) and then prepared for cGMP/cAMP quantification by competitive immunoassay kit. Cells treated with forskolin (5 μM), DEA-NO (1 μM) for 1 min, or IBMX (50 μM) for 10 min were used as positive controls. Data are presented as fold increase compared to control set to 1 (mean±S.E.M., n=5, *P<0.05 compared to control). (c) Washed human platelets were stimulated with ABT-737 (1 μM, 60 min), TQ (40 μM, 10 min), forskolin (5 μM, 1 min), DEA-NO (0.1 μM, 1 min) with or without preincubation with inhibitors of AC (SQ22536, 100 μM, 10 min) or GC (ODQ, 10 μM, 10 min). Samples were analysed by western blot for VASPSer239 phosphorylation. Actin blots served as loading control. Presented western blot results are representative of five independent experiments. (d) Washed human platelets (3 × 108/ml) were stimulated with ABT-737 (1 μM), TQ (40 μM), or Calyculin A (1 μM) for 60 min. Platelet lysates were then analysed by western blot with phospho-Ser/Thr-specific antibodies (upper panel) or P-VASP239-specific antibodies (lower panel). Data are representative of at least four independent experiments. (e) In vitro phosphatase assay: the PP1A/PP2A-driven release of free phosphate from a defined phosphopeptide after 15 min incubation (corresponding to 50% phosphate release of maximal phosphate release measured after 1 h incubation with PP1A or PP2A) was measured in the presence and absence (ø) of TQ or ABT-737, using a colorimetric reaction at 600 nm as described in the method part. Data are presented as optical density (OD) at 600 nm (mean±S.E.M., n=3; n.s. no significant difference compared to phosphatase reactions without TQ/ABT-737 supplementation)
