Halt protease inhibitor cocktail 100x

WT and KO sperms were collected from the cauda epididymis in PBS supplemented with 0.1% PVP and centrifuged at 3,000 g for 7 min. Pellets were snap-frozen in liquid nitrogen and kept at −80°C until analysis. The frozen pellets were re-suspended in Laemmli buffer (4X) and boiled for 10 min (lysis protocol #1) in a lysis buffer composed of 50 mM Tris HCl (pH8), 10 mM DTT, and 2% SDS boiled for 10 min with vigorous agitation (lysis protocol #2), in M-PERTM Mammalian Protein Extraction Reagent supplemented with HaltTM Protease Inhibitor Cocktail (100X) (Thermo Fisher Scientific) for 1 h at 4°C with vigorous agitation (lysis protocol #3) or RIPA buffer supplemented with HaltTM Protease Inhibitor Cocktail (100X) for 1 h at 4°C with vigorous agitation (lysis protocol #4). After incubation, the samples were centrifuged at 8,200 g for 10 min to discard cell debris. The supernatant containing sperm proteins was separated by SDS-PAGE and transferred to PVDF membranes which were probed with two custom polyclonal rabbit antibodies (anti-OLFR601-1 and anti-OLFR601-2, 1:1000 v/v in TBST 1X 1% BSA) before visualization by chemiluminescence (Pierce ECL-Plus, Thermo Fisher Scientific).

Cells were lysed in a lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton-X 100 and a Halt^TM Protease Inhibitor Cocktail (100x, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured by a Qubit^TM Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Equal amount of proteins was run on 10% Tris-tricine gel and blotted to polyvinylidene fluoride (PVDF) membrane with a Bio-Rad Wet Blotting System (Bio-Rad Hungary, Budapest, Hungary). The HER2 receptor was detected by an ErbB2 (HER2) antibody cocktail (Thermo Fisher Scientific, MA5-14057, produced in mouse, 1:500) and an anti-mouse-horseradish peroxidase (HRP) secondary antibody (Thermo Fisher Scientific, 32430, produced in goat, 1:500). As a loading control, β-actin was detected by an anti-actin antibody (Santa Cruz Biotechnology, Dallas, TA, USA, sc-1616, produced in goat, 1:2500) and an anti-goat-HRP secondary antibody (Santa Cruz Biotechnology, sc-2354, produced in mouse, 1:2000). After the addition of enhanced chemiluminescence (ECL) substrate (SuperSignal^TM West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific), the chemiluminescent signal was detected by ChemiDoc XRS+ Detection System (Bio-Rad Hungary).

Denaturing protein extraction was achieved from the three biological replicates of each cultivar×strain combination collected at 72 hpi using the TCA/acetone procedure as described in Bonhomme et al. (2012) (link). Protein solubilization was performed in an urea-thiourea buffer [6 M urea; 2 M thiourea; 100 mM ammonium bicarbonate; 1% Halt^TM Protease Inhibitor Cocktail 100X (Thermo Fisher Scientific 78429); 0.1% ProteaseMAX^TM Surfactant (Promega V2071)] by following the ratio 10 μL per mg of dry matter. Protein digestion were performed as described in Fabre et al. (2019) (link). Tandem mass spectrometry analyses were achieved using a nanoESI Q Exactive^TM HF-X Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer (Thermo Fisher Scientific 0726042) coupled with an Ultimate 3000 HPLC (Thermo Fisher Scientific). HPLC gradients and data acquisition parameters were set as described in Fabre et al. (2019) (link).