Journal: Cellular and Molecular Life Sciences: CMLS
Article Title: ELAPOR1 mediated vesicle traffic is required for acrosome biogenesis and male fertility in mice
doi: 10.1007/s00018-026-06125-0
Figure Lengend Snippet: ELAPOR1 was required for recruitment of clathrin onto the pro-AVs. ( A ) Schematic diagrams of flag tagged recombinant ELAPOR1. ( B ) Representative co-immunoprecipitation of recombinant ELAPOR1 with clathrin, AP1, AP2 and AP3 in HEK-293T cells. Western blot analysis showed the interaction between ELAPOR1 and clathrin as well as APs, while the relative targets detected on cell lyses were performed as loading control. ( C ) Immunofluorescent co-staining of clathrin (red) and TGN46 (green, trans-TGN marker) in spermatids. The Elapor1 cKO spermatids displayed rare co-localization of clathrin and TGN46. The arrow indicates the acrosome. Scale bar = 10 μm. ( D ) The localization of clathrin on pro-AVs analyzed by IEM showed that clathrin localized on the surface of most pro-AVs situated between the Golgi apparatus and the acrosome in normal spermatids, whereas clathrin on the pro-AVs disappeared when Elapor1 deficiency. G, Golgi apparatus; A, Acrosome; pre-A, Preacrosomal granules; red arrows, immunogold labelled clathrin. Scale bars in low-magnification = 200 nm, in high-magnification = 100 nm
Article Snippet: The unspecific binding sites were blocked with 5% goat serum in PBS and then incubated overnight at 4 °C with the primary antibodies against Elapor1 (1:200 dilution), ACRV1 (1:200 dilution, #14040-1-AP, Proteintech), GM130 (1:1000 dilution, #610822, BD Biosciences), Clathrin (1:200 dilution, #26523-1-AP, Proteintech), and TGN46 (1:200 dilution, #66477-1-Ig, Proteintech), respectively.
Techniques: Recombinant, Immunoprecipitation, Western Blot, Control, Staining, Marker
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