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. 2022 Aug 12;8(31):eabo6049. doi: 10.1126/sciadv.abo6049

Fig. 6. C11orf94 regulates formation of Izumo1 complexes.

Fig. 6.

(A) Analysis of Izumo1-containing complexes in the testis of Wt or C11orf94-deficient (C11 KO) mice. Homogenized testes were lysed and subjected to blue native PAGE with subsequent Western blotting. Monomeric (filled arrow head) Izumo1 and Izumo1 in HMW complexes (HMW) are highlighted. (B) Quantification of (A). N = 3, n = 5, two-tailed unpaired Student’s t test. (C) HEK293T cells were transfected with ACE3-HA alone or in combination with Izumo1-3xFLAG and C11orf94-3xmyc. Izumo1-3xFLAG was precipitated from CHAPSO homogenates of these cells using FLAG-specific beads, and proteins were detected in whole-cell lysates (lysate) or immunoprecipitated (IP) samples. Asterisk, antibody band. (D) Quantification of (C). N = 2, n = 5, two-tailed unpaired Student’s t test. (E) Interaction of ACE3-HA with C11orf94-3xFLAG, SPPL2c-3xFLAG, or Izumo1-3xFLAG was analyzed by coimmunoprecipitation experiments using CHAPSO lysates of transfected HEK293T cells and anti-FLAG beads. Asterisk, antibody band. (F) Izumo1 was precipitated from CHAPSO lysates of noncapacitated sperm cells from either Wt or C11 KO mice, and lysates and IP samples were analyzed by Western blotting. Actin served as loading control. Asterisk, antibody band. (G) Quantification of (F). N = 1, n = 3, unpaired Student’s t test. *P ≤ 0.05.