Figure 3. E. carinatus venom stimulates in vivo NETosis.

(a) Representative western blot of the time course of H3Cit and MPO appearance in E. carinatus venom (LD₅₀)-injected mouse tail tissue (left) and quantification of the H3Cit levels compared with the H3 levels (top, right) and MPO level compared to GAPDH (bottom, right). AU, arbitrary units; H3Cit, citrullinated histone 3; H3, histone 3; MPO, myeloperoxidase. The data are presented as mean±s.e.m; Student's t-test, ***P<0.001 versus control; n=4 for the control, 2, 4, 8 and 16 h samples; n=5 for 24 h, day 3 and day 10 samples. The PVDF membranes were cut based on molecular weight of respective protein using protein molecular weight marker and then probed with respective antibodies. (b) Representative immunofluorescence images of mouse tail tissue 8 h after E. carinatus venom (LD₅₀) injection, focused beneath the epithelial layer. Scale bar, 100 μm (n=4). (c) Representative confocal image of E. carinatus venom (LD₅₀)-injected mouse tail tissues focused beneath the epithelial layer. The area enclosed by the yellow box is magnified and shown on the right. Scale bars, 100 μm (left), 50 μm (right); n=3.