Forskolin induces dephosphorylation of AnxA2. (A) HUVECs were incubated in forskolin/IBMX-containing stimulation medium or basal medium containing DMSO as vehicle control for 30 minutes, and PNSs were subjected to immunoprecipitation (IP) using anti–phosphoserine-coupled agarose. The amount of AnxA2 present in the PNS and the immunocomplexes was quantified by immunoblotting using mouse monoclonal anti-AnxA2 antibodies (clone HH7). One representative example is shown on the left. For quantification, AnxA2 levels in PNS fractions (input) were normalized to the respective level of the Ig heavy chain signals (internal loading control) and calculated as percentage of protein precipitated from unstimulated cells (right). Statistical significance of the results obtained from 3 independent experiments was evaluated by unpaired Student t test. (B) Serum-starved HUVECs were pretreated for 5 hours with 50 nM FK-506 or DMSO as vehicle control at 37°C. Subsequently, cells were incubated in forskolin/IBMX-containing stimulation or basal medium for 30 minutes, and PNSs were subjected to anti–phospho-serine IP as described in A. PNS and IP fractions were probed for AnxA2, and a representative immunoblot of 2 independent experiments is shown. Numbers below the blot indicate AnxA2 levels in the IPs that were calculated as percent of protein precipitated from unstimulated cells as described in A. (C) HUVECs were preincubated in medium containing FK-506 or DMSO (vehicle control) and then subjected to forskolin/IBMX treatment as outlined in Methods. The amount of VWF released into the cell culture supernatant was analyzed by ELISA, and statistical significance of the results obtained from 11 independent experiments was evaluated by unpaired Student t test. Bars represent mean ± SEM. The basal secretion is not affected by FK-506 treatment, whereas the calcineurin inhibitor significantly reduces the forskolin-induced secretion.