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. 2022 Jun 3;10:854115. doi: 10.3389/fcell.2022.854115

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Copyright © 2022 L, JG, I, M and P.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Expression of recombinant TMEM95 and pull-down. (A) Schematic representation of recombinant TMEM95 proteins. FLAG sequence (), histidine tail (), and transmembrane domain () indicated. (B) Proteins were expressed in CHO cells, separated by SDS-PAGE and analyzed by Western blot. TMEM95 and TMEM95∆Tm were probed with anti-TMEM95 and anti-FLAG antibodies. (C) SDS-PAGE and Western blot of BTMEM95∆Tm. Input: medium with secreted TMEM95∆Tm before conjugation. Output: media after conjugation. IP:Ni+2 Beads: eluted fraction containing TMEM95∆Tm. (D) Venn diagram from the listed sperm proteins identified by HPLC-MS/MS analysis. Proteins from the sperm lysate that bind non-specifically to Sepharose® beads (pink), proteins eluted from BTMEM95∆Tm (blue), and specifically bound to TMEM95∆Tm (i.e., sperm proteins interacting with TMEM95, green, Supplementary Table S1).

Inline graphic — Expression of recombinant TMEM95 and pull-down. (A) Schematic representation of recombinant TMEM95 proteins. FLAG sequence (), histidine tail (), and transmembrane domain () indicated. (B) Proteins were expressed in CHO cells, separated by SDS-PAGE and analyzed by Western blot. TMEM95 and TMEM95∆Tm were probed with anti-TMEM95 and anti-FLAG antibodies. (C) SDS-PAGE and Western blot of BTMEM95∆Tm. Input: medium with secreted TMEM95∆Tm before conjugation. Output: media after conjugation. IP:Ni+2 Beads: eluted fraction containing TMEM95∆Tm. (D) Venn diagram from the listed sperm proteins identified by HPLC-MS/MS analysis. Proteins from the sperm lysate that bind non-specifically to Sepharose® beads (pink), proteins eluted from BTMEM95∆Tm (blue), and specifically bound to TMEM95∆Tm (i.e., sperm proteins interacting with TMEM95, green, Supplementary Table S1).