The validated antibodies were then used to probe Western blots of sucrose gradient fractions obtained as described in Materials and Methods (Fig. 2) . Among the assayed proteins, it was possible to distinguish three different gradient distributions. Although all of them were found in the light fraction used previously for tandem mass spectrometry identification [20] , TEX101, SPAM1, FLOT1, and FLOT2 were present exclusively in the light fractions; IZUMO was more widespread and was also found in intermediate-density fractions; and a third set of proteins comprising SLC2A3, BSG, HK1, and CAV2 was found in most fractions, including the heavy ones. ADCY10 distributed exclusively in the heavy fractions.