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. 2014 Jan 2;10(1):e1003858. doi: 10.1371/journal.ppat.1003858

Figure 3. M2 activation of the NFAT pathway is CsA and FK506 sensitive and requires extracellular calcium flux.

Figure 3

(A, B and C) Impact of inhibitors on M2-induced NFAT activity. The NFAT reporter assay was performed as in Figure 2A.Doxycycline was added for 48 hours. CsA (500 ng/mL), FK506(1 µM), EGTA(2.5 mM), BAPTA-AM(5 µM), PP2 (10 µM) and PP3 (10 µM) were added 24 hours post-nucleofection and the lysates were assayed for luciferase activity at 48 h post-nucleofection. Data are represented as fold over respective uninduced conditions, setting the uninduced samples in each drug treatment to 1. (D) The indicated cell lines were either left uninduced or induced with doxycycline for 48 hours. Whole cell lysates were harvested and 60 µg protein was resolved by SDS-PAGE followed by immunoblotting for NFATc1 using antibodies mentioned in materials and methods. β-actin was used as a loading control.