Fig. 6: Identification of EFCAB5 as a sperm axoneme-specific RS3 protein.

a A schematic drawing of the application of BioID2 in mature spermatozoa. Spermatozoa from WT and Cfap91^-/- TG males were incubated in a medium supplemented with biotin for 16 h. Collected spermatozoa were lysed, and biotinylated proteins were pulled down by streptavidin (SA). b Immunoblotting was performed after the pull-down of biotinylated proteins in WT and Cfap91^-/- TG males. EFCAB5 was not detected in the input, likely due to low protein abundance and/or low solubilization efficiency (0.4% SDS compared to 1% SDS or 6 M urea in other experiments). IZUMO1 served as a loading control for inputs. c Molecular weight and total spectra of twenty-four proteins only identified in Cfap91 KO TG spermatozoa. CFAP91 and EFCAB5 were identified with the most peptides among all twenty-four proteins. A linear regression line with 95% confidence bands is shown. Slope = 0.06 and Y-intercept = 5.38. d Venn diagram of CFAP91 immunoprecipitates, proximity labeling-identified proteins, and mammalian sperm RS3 proteins. e RT-PCR of Efcab5 utilizing cDNA from multiple mouse organs. Actb was used as a loading control. f Immunoblot analyses of human spermatozoa. g immunoblotting using testes and cauda epididymal spermatozoa from WT and Cfap91^-/- males with an anti-EFCAB5 antibody. Alpha-tubulin (α-TUB) served as a loading control. h Fractionation using WT spermatozoa. Signals of EFCAB5, LRRC23, and CFAP251 were found in the SDS-soluble fraction.