FIG 4.

Confirmation of inhibitory activity of NPP using the SNAG construct (GST–SNAP-25–GFP). SNAG is labeled as the negative control (NC; lane 1). SNAG incubated with the 2 nM LCA served as the positive control (PC; lane 2). LCA was incubated with serially diluted (2×) inhibitor (80, 40, 20, 10, 5, 2.5, and 1.25 μM; lanes 4 to 10). Percent inhibition was determined using densitometry analysis with Bio-Rad Image Lab (v.5.2.1) software. SNAP-25 cleavage by NPP was reduced in a concentration-dependent manner. The IC₅₀ was determined to be 6.3 ± 2.02 µM (mean ± SD, n =3). The gel is representative of the gels from 3 independent experiments.