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Fig. 1 | BMC Biology

Fig. 1

From: GLIPR1L1 is an IZUMO-binding protein required for optimal fertilization in the mouse

Fig. 1

Identification of mouse sperm multimeric protein complexes with affinity for homologous oolemmal proteins. a Mouse spermatozoa were purified under non-capacitating (Non-Cap) or capacitating (Cap) conditions. A portion of the latter population was also challenged with A23187 to induce the acrosome reaction (AR). To detect native protein complexes with affinity for oolemmal proteins, far-western blotting with biotin-labeled preparations of oocyte lysates (Far-Western) was undertaken. Four predominant oolemmal protein-binding complexes (arrowheads, I–IV) were identified. Each experiment was replicated a minimum of three times and representative gels and blots are shown. The numbers on the left correspond to the molecular weight (kDa) of native PAGE protein standards. b Validation of GLIPR1L1 and IZUMO1 antibodies. The specificity of the antibodies used in this study was confirmed by immunoblotting against sperm protein extracts. This experiment was replicated three times and immunoblots are shown. The numbers on the left correspond to the molecular weight of the protein standards. c Identification of mouse sperm protein complexes comprising IZUMO1 and GLIPR1L1. Populations of non-capacitated (Non-Cap), capacitated (Cap), and acrosome-reacted (AR) mouse spermatozoa were solubilized in blue native lysis buffer. The extracted proteins were resolved on BN-PAGE gels before being prepared for immunoblotting with either IZUMO1 or GLIPR1L1 antibodies. Arrowheads indicate the predominant complexes recognized by each antibody. Red arrowheads correspond to complexes (I and IV) that co-migrated with those that bound oolemmal proteins (see Fig. 1a). Each experiment was replicated a minimum of three times and representative images are shown. The numbers on the left correspond to the molecular weight (kDa) of native PAGE protein standards

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