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

Fig. 1. C11orf94 is a testis-specific type II ER protein.

Fig. 1.

(A) Scheme of the wild-type C11orf94 expression construct equipped with a C-terminal 3xFLAG-tag. At the N terminus of the protein, a predicted TMD, which might also function as signal peptide (SP), is highlighted. (B) HeLa cells were transfected with mC11orf94-3xFLAG and analyzed by indirect immunofluorescence. Scale bars, 10 μm. (C) HEK cells were transfected with mC11orf94-3xFLAG, and cellular membranes were purified by ultracentrifugation. C11orf94 was detected by Western blotting; the membrane protein LIMP-2 and the cytosolic EEF-2 protein served as controls. (D) An artificial N- or C-terminal N-glycosylation site (Nsite, sequence NIS) was introduced to C11orf94. HEK cells transfected with the indicated constructs were lysed, and lysates were subsequently subjected to endoglycosidase H (EndoH) treatment. LIMP-2, an endogenously glycosylated protein, served as control. (E) Sperm and testis lysates of wild-type (Wt) or C11orf94-deficient (C11 KO) mice were analyzed by Western blotting. (F) Expression of C11orf94 within the testis was visualized by IHC using a C11orf94-targeting antibody. Asterisk, unspecific staining. Scale bars, 25 μm. (G) Expression of C11orf94 in different spermatid stages was evaluated by staining of testis cryosections from Wt mice with anti-C11orf94 and PNA-FITC as acrosomal marker. Scale bars, 5 μm.