Fig 2: ATP12A and PNA localization in sperm cells from cattle. Representative fluorescence microscope images showing immunodetection of ATP12A (green) and TRITC-conjugated PNA (red) in frozen/thawed sperm cells. The secondary antibody for ATP12A was conjugated to Alexa Fluor 488. Nuclei were counterstained with DAPI (blue). Insets in the four panels represent 4× magnification of the same representative sperm cell as indicated by the white arrows. Scale bar: 10 µm.

Fig 3: ATP12A protein expression in sperm cells from cattle, buffalo and sheep. Representative fluorescence microscope images showing immunodetection of ATP12A (green) in frozen/thawed sperm cells. The secondary antibody was conjugated to Alexa Fluor 488. Nuclei were counterstained with DAPI (blue). Insets in the merge panels represent 2× magnification of representative sperm cells indicated by white arrows. Yellow arrows within the green channel indicate positive staining of ATP12A at the flagellum. Scale bar: 20 µm.

Fig 4: Western blot of sperm homogenates from cattle, buffalo and sheep samples. Immunocomplexes obtained using the EpiGentek A62134 antibody against ATP12A and an anti-rabbit secondary antibody (Merck/MilliporeSigma) conjugated to peroxidase were detected at about 100 kDa using the ECL plus reagent (GE Healthcare Life Sciences; see Materials and Methods for details).

Fig 5: The lateral views of 3D crystallized structures of bovine ATP1A1 in complex with ATP1B1 and of swine ATP4A in complex with ATP4B are reported in green/cyan and yellow/pink cartoon representation, respectively. The 3D comparative models of the bovine ATP12A (white cartoon) in complex with ATP1B1 (cyan cartoon) or ATP4B (orange cartoon) are also reported in cartoon representation. calculated interaction energies are reported.