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. 2014 Dec 30;9(12):e116423. doi: 10.1371/journal.pone.0116423

Figure 10. Alterations in expression and activation of NOS in ChEC.

Figure 10

A: The level phosphorylated-eNOS (p-eNOS) and total eNOS, iNOS, and nNOS (not shown) in cell lysates were analyzed by Western blotting. The β-actin was used for loading control. Please note a significant increase in the level of p-eNOS and iNOS in TSP1−/− ChEC compared with TSP1+/+ cells (*P<0.05; n = 3). This was confirmed by measuring band intensities relative to β-actin, and we did not detect nNOS in both cell types (not shown). B: intracellular nitric oxide (NO) level in ChEC was measured using 4-amino-5- methylamino-2,7-difluorofluorescein (DAF-FM) as described in Methods. Please note a significant increase in intracellular NO level in TSP1−/− ChEC compared with TSP1+/+ cells (*P<0.05; n = 3). C: secreted level of VEGF in ChEC was determined using an ELISA immunoassay as described in Methods. Please note the similar level of VEGF secretion in ChEC (P>0.05; n = 3). These experiments were repeated with two different isolations of cells with similar results.