Protein preparation and western blot

Total proteins were extracted from sperm, testis, and cultured mammalian cells as previously described (Hwang et al., 2019). In short, collected epididymal sperm cells were washed with PBS and lysed in 2× LDS sampling buffer for 10 min at room temperature with agitation (RT). The whole sperm lysates were centrifuged at 14,000 × g for 10 min at 4°C. Testes were homogenized in 0.32M sucrose and centrifuged at 1000 × g for 10 min at 4°C to remove cell debris and nuclei. 1% Triton X-100 in PBS containing protease inhibitor cocktail (cOmplete, EDTA-free, Roche) was added to the cleared homogenates to make total testis lysate. The lysates were centrifuged at 4°C, 14,000 × g for 30 min and the supernatant was used for the downstream experiments. Transfected HEK29T cells and COS-7 cells were washed and lysed with 1% Triton X-100 in PBS with protease inhibitor cocktail (Roche) at 4°C for 1 hr. Cell lysates were centrifuged at 14,000 × g for 30 min. All the solubilized protein lysates from the sources described above were reduced by adding dithiothreitol (DTT) to 50 mM and denature by heating at 75°C for 5 min (testis and cultured cells) or 10 min (sperm).

Discontinuous sucrose density gradient centrifugation was performed as previously described (Kaneto et al., 2008). To isolate and solubilize membrane fraction without using a detergent, cauda epididymal sperm cells washed and suspended in PBS (1.0 × 10⁸ cells/mL) were sonicated 3 times for 1 s each. Sonicated sperm cells were then centrifuged at 5000 × g for 10 min at 4°C and the solubilized membrane fraction in the supernatant was collected. The solubilized membrane fraction was pelleted by ultracentrifugation at 100,000 × g for 1 hr at 4°C and resuspended with PBS. The membrane suspension was mixed with an equal volume of 80% sucrose in PBS. A discontinuous sucrose gradient was layered with the 40%, 30%, and 5% sucrose solution from bottom to top in a tube discontinuously. The gradient was ultracentrifuged at 200,000 × g for 20 hr at 2°C. Proteins collected from each fraction were precipitated with 5% of trichloroacetic acid, ethanol washed, and dissolved in SDS sampling buffer.

Sperm membrane fractions from 1 × 10⁶ sperm cells prepared as above were treated with protein phosphatase 1, (PP1, 0.1 unit; NEB), protein tyrosine phosphatase (PTP1B, 5 units; Abcam), or sodium orthovanadate (Na₃VO₄, 1 mM; NEB) to test dephosphorylation of CatSper1. The membrane fractions were incubated with the phosphatases or Na₃VO₄ in a reaction buffer containing 20 mM HEPES, 0.1 mM EDTA, and 0.1 mM DTT at 30°C for the indicated times. The isolated sperm membrane was solubilized by adding Triton X-100 to the final 0.1% in PBS (PBS-T) for the indicated times at RT.

Glycosylation of CatSper1 from cauda sperm was tested using PNGase F (Sigma-Aldrich) and O-glycosidase (NEB) according to the manufacturer’s instructions. Sperm cells (4 × 10⁶ cells) were washed with 1× reaction buffer for each enzyme by centrifugation at 800 × g for 3 min. Sperm pellets were re-suspended with each 1× reaction buffer (20 mM or 50 mM sodium phosphate, pH 7.5 for PNGase F and O-glycosidase, respectively) and followed by sonication and centrifugation to collect sperm membrane fraction as described above. Collected supernatants were incubated with denaturation buffer at 100°C for 5 min to denature glycoproteins before subject to enzymatic deglycosylation. The denatured sperm membrane fractions were incubated with detergent buffer (0.75% IGEPAL CA for PNGase F; 1% NP-40 for O-glycosidase) and each glycosidase (PNGase F, 2 nM unit; O-glycosidase, 80,000 unit) at 37°C for 1 hr. All the enzyme-treated samples were mixed with LDS sampling buffer and denatured after adding DTT to 50 mM at 75°C for 2 min.

Proteolysis of the recombinant CatSper1 protein by sperm lysate was performed as previously described (Chung et al., 2014). Solubilized recombinant FL-CatSper1 and ND-CatSper1 were pulled-down with anti-HA agarose (EMD Millipore) for 1 hr at RT. The enriched recombinant proteins were incubated with 30 μL of sperm lysates solubilized from 3.0 × 10⁵ sperm cells at 37°C for the indicated times. Sperm lysates were prepared by sonication and incubation in PBS-T without protease inhibitor at 4°C for 1 hr. After incubation, the mixture of recombinant protein and sperm lysates were mixed to 2× LDS sampling buffer and denatured by adding DTT to 50 mM at 75°C for 10 min.

Denatured protein samples were subjected to SDS-PAGE. Rabbit polyclonal CatSper1 (2 μg/mL), CatSper3 (2 μg/mL), CatSperε (1.6 μg/mL), and CAIV (1:500) antibodies and monoclonal HA (clone C29F4; 1:2,000), caveolin1 (clone 2297, 1:500), acetylated tubulin (clone 6-11B-1; 1:20,000), and phosphotyrosine (clone 4G10; 1:1000) antibodies were used for western blot. Anti-mouse IgG-HRP (1:10,000) and anti-rabbit IgG-HRP (1:10,000) were used for secondary antibodies.