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. 2023 Feb 14;120(8):e2207263120. doi: 10.1073/pnas.2207263120

Fig. 4.

Fig. 4.

1700029I15Rik Interacts with Proteins Involved in N-glycosylation, Disulfide Bond Formation, and Vesicular Trafficking and Facilitates the Expression of Multiple Acrosomal Membrane Proteins. (A) Venn diagram depicting shared and unique protein hits identified in three biological replicates of co-IP/MS experiments. Only the proteins specifically detected in the wild-type samples are included. The proteins detected in at least two replicates were subjected to STRING analysis (20, 21). For each replicate, 1700029I15Rik and its interacting proteins were coimmunoprecipitated from wild-type and knockout testis lysates using antibody-crosslinked agarose resin. (B) GO and KEGG analyses of 1700029I15Rik-interacting proteins. Functional annotations were conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (22). Only the 31 proteins concurrently detected in at least two replicates of the co-IP/MS experiments were analyzed. (C and D) In vitro validation of protein–protein interactions. 1700029I15Rik and its associated proteins were coimmunoprecipitated from the lysates of HEK293T cells transiently expressing 1700029I15Rik-3 × FLAG using antibody-conjugated magnetic beads. The co-IP products were subjected to SDS-PAGE and Western blot analyses to validate protein–protein interactions. (E) Western blot analyses of OSTC, PDIA3, RPN2, and TMED10 in wild-type and knockout testes. BASIGIN was analyzed in parallel as a loading control. (F and G) Western blot analyses of various proteins in wild-type and knockout testes. Unless specified otherwise, all protein samples were processed under reducing and denaturing conditions. NR, non-reducing and non-denaturing. The DET fractions of wild-type and knockout testis proteins, extracted by Triton X-114, were used for immunodetection of SPACA6. (H) Western blot analyses of various membrane proteins in wild-type and knockout sperm. (I) Western blot detection of SPACA6 in the sperm of wild-type, Izumo1–/–, Fimp–/–, Sof1–/–, Tmem95–/–, Dcst1/2–/–,1700029I15Rik–/–, and Spaca6–/– male mice. BASIGIN was analyzed as a loading control.