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. 2019 Mar 1;9:3312. doi: 10.1038/s41598-019-39842-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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SDS-PAGE and Western blot analysis of mice urine samples. Urine of mice treated daily with vehicle saline or crotamine, by intraperitoneal (ip) route 1.5 h before urine collection, were applied to a 15% SDS-PAGE before the (a) staining with Coomassie blue, (b) silver staining, or (c) Western blot analysis with anti-crotamine antibody followed by development with alkaline phosphatase substrates NBT/BCIP. The presence of a protein band of about 5 kDa, corresponding to the full-length crotamine, is indicated by the arrows. MW: molecular weight markers (kDa), lane 1: native crotamine (100 ng), lane 2: urine of drug-naïve mouse receiving a single injection of vehicle saline (100 µL/animal), lane 3: urine of drug-naïve mouse receiving a single injection of native crotamine (30 µg/100 µL/animal), lane 4: urine of negative control mice treated daily with vehicle (100 µL/animal) for 21 days, and lane 5: urine of mice treated daily with crotamine (1 μg/100 µL/animal) for 21 days. In each lane, 5 µL of urine sample were loaded. The gels (a and b) and blot (c) are cropped along the edges. Complete uncropped blots/gels are presented in Supplementary Figure 3.