Calyculin A 花萼海绵体诱癌素A

体外研究：

细胞类型（Cell type）：Platelet-rich plasma (PRP) sample

药物配制（Preparation）：Calyculin-A (CLA) was dissolved in DMSO

实验方法（Assay）：PRP (110μL) was preincubated with either HEPES buffer containing 0.5% DMSO as control or the protein phosphatase inhibitor CLA (50nM), for 30 minutes at 37°C in a water bath. After preincubation, platelets were activated either by TRAP (20μM) or by thiomersal (200mM) for 15 minutes at 37°C in a water bath. Then PRP (5μL) was stained with 5μL monoclonal CD41-PE antibody and 5μL Annexin-V-FITC and Annexin-V binding buffer (1x concentrate) was added to examine the PS-expression of the platelets.

体外研究：

细胞类型（Cell type）：Xenopus Oocytes

药物配制（Preparation）：Calyculin A was prepared in 100% ethanol as a 1 mM stock solution. Immediately after dilution in distilled water to the appropriate concentration, calyculin A was microinjected into the oocytes according to the RNA injection protocol.

实验方法（Assay）：The NMDA-induced currents of striatal mRNA-injected oocytes were measured before and 8 min after 1-min application of 50 μM forskolin or before and 12 min after 1-min application of 10 nM PMA. The same protocol was followed after injecting oocytes with calyculin A to a final intracellular concentration of 500 nM 60–90 min before voltage-clamping. The intracellular concentration of calyculin A was calculated by assuming standard oocytes with a volume of 0.5 μl.

体外研究：

细胞类型（Cell type）：Living crane-fly spermatocytes

药物配制（Preparation）：Calyculin A was dissolved in DMSO as a 1mM or 50 μM stock solution.

实验方法（Assay）：Living crane-fly spermatocytes were perfused with insect Ringer’s solution or with Calyculin A in insect Ringer’s solution, at final concentrations of 0.5 μM, 100 nM, 50 nM, 20 nM, 10 nM or 5 nM, prepared from a 1 mM or a 50 μM Calyculin A stock in DMSO. Results showed that concentrations of 5 nM and 10 nM had no effect on anaphase chromosome movement, 20 nM had inconsistent effects, and 50 nM had consistent effects. Thus we used 50 nM for most of the experiments.