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Fig. 1 | BMC Biology

Fig. 1

From: Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions

Fig. 1

Mouse epididymosomes mediate the transfer of biotinylated proteins to homologous spermatozoa. a Epididymosomes were labeled with membrane impermeant or membrane permeant biotin reagents (i.e., sulfo-NHS-LC-biotin and BMCC-biotin, respectively) before being co-cultured with spermatozoa in vitro for 1 h. After incubation, spermatozoa were solubilized and lysates prepared for Western blotting and affinity labeling with HRP-conjugated streptavidin to assess the incorporation of biotinylated epididymosome cargo into their proteome. To demonstrate the selectivity of the transfer process, equivalent lysates from naïve populations of sperm that remained unexposed to epididymosomes were resolved alongside the treatment groups (Sperm only), revealing minimal endogenously biotinylated proteins. Blots were stripped and re-probed with α-tubulin to confirm an equal quantity of protein was loaded in each lane. b Unlabeled populations of epididymosomes (ES only) were also subjected to Western blotting, revealing no endogenously biotinylated proteins. The presence of proteins in this blot was affirmed by re-probing with the epididymosome marker FLOT1. These experiments were repeated three times and representative blots are shown

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