Protein marker 6

Electrophoresis was performed using the general principle [38 ] and Omni Page electroblotter (Cleaver Scientific). OTf PC2 of the test samples was diluted to a final concentration of 2 mg/mL protein, using 2-mercaptoethanol Laemmli buffer (Sigma Aldrich) and bromophenol blue (Sigma Aldrich). After incubating the samples for 10 minutes at 96°C, 5 μL of each sample was loaded on a polyacrylamide migration gel of 10% and a concentration gel of 4%. A molecular marker, Protein Marker VI (AppliChem), containing a mixture of 12 proteins with molecular weight ranging from 10 to 245 kDa, was also run on the gel. Electrophoresis was performed at 90 mV and 185 mA, for 90 minutes, and staining was performed with Coommassie Brilliant Blue R250 (Sigma Aldrich).

Sperm flagella and heads from S. purpuratus, CHO K1 cells stably expressing HA-tagged SpSLC9C1 (SpSLC9C1-HA), and CHO K1 control cells (mock) were lysed by sonification in a hypotonic buffer containing 10 mM Hepes/NaOH, pH 7.4, 2 mM EDTA, and protease inhibitor mixture mPIC (Sigma Aldrich, St. Louis, USA). The suspension was centrifuged for 10 min at 500 × g. The supernatant was used for Western blotting. Twenty µg protein per lane were separated by SDS-PAGE using a precast NuPage Novex 7% Tris-Acetat Protein Gel (Thermo Fisher Scientific, Waltham, USA). The samples were heated for 5 min at 95 °C prior to separation. Protein Marker VI (AppliChem, Darmstadt, Germany) and HiMark Pre-Stained Protein Standard (Thermo Fisher Scientific) were used as molecular weight markers. Proteins were transferred onto an Immobilon FL PVDF membrane (Merck Millipore, Darmstadt, Germany), probed with antibodies, and analyzed using the Odyssey Imaging System (LI-COR, Bad Homburg, Germany). All figure panels were taken from the same western blot. Figures were prepared using CorelDrawX6 and Photo-PaintX6 software (both from Corel Corporation). Primary antibodies were: SpSLC9C1-SU2 (1:3000), rat-anti-HA (1:3000; Roche Applied Science catalog no. 11867431001, Penzberg, Germany). Secondary antibodies were as follows: IRDye680 and IR800 antibodies (LI-COR, 1:25,000).