Alliance 2

The DNase activity of the venom was determined by agarose gel electrophoresis/DNase radial diffusion assay. Briefly, 750 ng of calf thymus DNA was independently incubated with the N. naja (5–50 μg ml⁻¹) venom for 60 min, at 37 °C in a final volume of 50 μl PBS. The reaction mixture was subjected to electrophoresis on 0.8% agarose gels at 50 V in TAE buffer (40 mM Tris-base and 1 mM EDTA, pH 8.0) for 1 h. Calf thymus DNA that had been treated with DNase 1 (5 U) served as a positive control. After electrophoresis, the gel was visualized and photographed on a ultraviolet transilluminator (Alliance 2.7, Uvitec). The N. naja venom DNase activity was inhibited by incubating the sample with actin (5–25 μM) for 10 min at 37 °C. Image has been cropped for presentation. Uncropped image is presented in Supplementary Fig. 11.
For radial diffusion assay, molten agarose (5 ml, 2.1%) containing herring sperm DNA (1 mg ml⁻¹) and ethidium bromide (1 μg ml⁻¹) was poured into wells of six-well culture plates and allowed to solidify. Seven-millimetre diameter wells were created at the centre of the dishes and loaded with E. carinatus venom (50–500 μg per well) for 24 h at 37 °C. DNase 1 (10 U) served as the positive control. After incubation, the plates were visualized and photographed on a ultraviolet transilluminator (Alliance 2.7, Uvitec).

Heart samples were
homogenized and solubilized in sodium dodecyl sulfate (SDS) buffer.
Protein quantification was performed using Qubit Proteinassay Kit
(Life Technologies, Monza, Italy) according to the manufacturer’s
instructions. Protein samples were mixed with a nonreducing and reducing
buffer and incubated for 5 min at either room temperature (reducing
and nondenaturating conditions) or 95 °C (reducing and denaturating
conditions). All samples were subsequently separated on a 10% gel
in SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham,
Euroclone, Italy). The membrane was blocked for 1 h with 5% nonfat
milk in the TBS containing 0.5% (v/v) Triton X-100. (Sigma-Aldrich)
and incubated overnight with polyclonal goat antibodies against NGAL
(1:500, Abcam, Prodotti Gianni, Milan, Italy). Blots were developed
using the Super Signal West Femto ECL substrate (Pierce, Euroclone,
Italy). Blot image acquisition was performed using Alliance 2.7 (UVITEC,
Eppendorf, Italy) and software Alliance 2.7 1D fully automated.

Tissue homogenates were standardized to DNA in SDS-PAGE sample buffer, as described 20 (link),21 (link), and separated by PAGE on a 4–12% BisTris gradient SDS gel and blotted onto polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA). Each gel had the same positive control (20 μg protein of 1% O₂ hypoxia-treated Lewis Lung whole-cell lysate) to normalize the signal between blots. Membranes were probed with primary antibodies against mouse HIF-1α (1/800; R&D Systems, Minneapolis, MN), GLUT-1 (1/20000; Abcam, Cambridge, UK), CA-IX (1/800, R&D Systems), or β-actin (1/10000, R&D Systems), and with secondary anti-goat or anti-mouse horseradish peroxidase-conjugated antibodies (DAKO, North Sydney, Australia). Membranes were developed using Amersham™ ECL Plus Western blotting detection system (GE Healthcare, Auckland, New Zealand), and protein bands were detected using a digital gel-doc system (Uvitec, Cambridge, UK). Multiple exposure times were used to ensure that the signal was not saturated, and measurements were taken in the linear range. Proteins bands were quantified using Alliance 2.7 software (Uvitec) and the protein of interest normalized against the positive control following confirmation of equal loading using β-actin.