Figure 7.

SubAB inhibits thrombin-induced Ca²⁺ release and entry in EC. HPAEC grown to confluence on 25 mm coverslips were treated with 0.1 µg/ml of SubAB or SubA or its inactive mutant SubA_A272B for 3 h. Cells were then loaded with Fura2-AM for 30 min and washed twice with Ca²⁺ free HBSS buffer and mounted on an inverted microscope. Calcium release from the intracellular stores was determined by perfusing Ca²⁺ free imaging buffer and stimulating cells with thrombin (2.5 U/ml). Store-operated Ca²⁺ entry was measured following addition of 1.26 mM Ca²⁺ (A,B). HPAEC treated with or without SubAB were loaded with Fura2-AM and stimulated with 30 µM CPA in the absence of extracellular Ca²⁺, to deplete ER Ca²⁺. This was followed by re-addition of 1.26 mM Ca²⁺ to determine Ca²⁺ entry (C,D). Fura-2 ratio (F-ratio 340/380) was calculated and analyzed with NIS-Element AR 3.0 Software. CPA, is cyclopiazonic acid.