Figure 2.

ET-1 induces activation of calcineurin/NFATc4 signaling pathway. (a) Representative traces demonstrating the effect of ET-1 (100 nM) on intracellular calcium concentration ([Ca²⁺]i) in PASMCs. (b) PASMCs were stimulated with 100 nM ET-1 for 30 min, calcineurin activity was determined using a calcineurin assay kit (n = 4 each group). (c) PASMCs were incubated with 100 nM ET-1 for 3 h, phosphorylation of NFATc4 was measured by immunoblotting, β-actin served as a loading control (n = 4 each group). (d) PASMCs were incubated with 100 nM ET-1 for 3 h, subcellular localization of NFATc4 was determined using immunofluorescence staining. Cells were stained with a primary antibody for NFATc4 and an Alexa Fluor 488-conjugated secondary antibody. Cell nuclei were stained with DAPI. (e) PASMCs were incubated with 100 nM ET-1 for 3 h, protein level of NFATc4 in the cytoplasmic and nuclear fractions were determined with immunoblotting. Lamin B and β-actin served as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 4 each group). *P < 0.05 versus control.