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. 2021 Oct 25;11:20979. doi: 10.1038/s41598-021-00494-1

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Effect of GSNO, l-NAME and AG on PKA substrates (PKAs-P) and tyrosine phosphorylation (Tyr-P). Sperm were incubated for 4 h under capacitating conditions in the absence of any treatments (CONTROL) or in the presence of GSNO, a NO donor, and l-NAME and AG (both NOS inhibitors). (a,b) Sperm protein extracts (n = 7) were analyzed for phosphorylation by Western blot using anti-PKAs-P or anti-Tyr-P as first antibodies, respectively. (c) β-tubulin (β-TUB) was used as a protein loading control. For signal quantification, each lane was normalized to its β-TUB optical density value. (d–g) Relative amount of signal quantified in each membrane using ImageQuant TL v8.1 software for PKAs-P and Tyr-P, respectively. Different letters (a, b, c) indicate statistically significant differences (P < 0.05) between groups. Images (a–c) were cropped from the corresponding blot showed in Supplementary Fig. S1 (lanes 3–6).