Aprotinin

The assays were performed in 96-well plates in a total reaction volume of 100 µL using 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20, 0.01% BSA for dilution of all samples. 5 nM purified active recombinant HATL5 or matriptase serine protease domain was incubated at 37°C for 60 min with 100 µm of the synthetic peptide L-1720 Suc-Ala-Ala-Pro-Arg-pNA (Bachem, Bubendorf, Switzerland) in the absence or presence (inhibitor and substrate added concomitantly) of HAI-1 (60 nM) (R&D, Minneapolis, MN), HAI-2 (40 nM) (R&D, Minneapolis, MN), aprotinin (2 µm), leupeptin (20 µm), benzamidine (2 mM), or serpinA1 (60 nM) (all from Thermo Scientific, Waltham, MA). Changes in absorbance at 405 nm were monitored using a Magellan NanoQuant Infinite M200 Pro plate reader (Tecan US, Inc., Morrisville, NC).

Where applicable, consumables were of electrophoresis grade or higher. Vacutainer^® SST (serum-separator tubes) and 21G butterfly needles were from BD (Franklin Lakes, NJ, United States). ReadyStrip^TM immobilized pH gradient (IPG) strips (17 cm, pH 3–10 non-linear), Bio-Lyte carrier ampholytes (pH 3-10, pH 4-6), and 2-D SDS-PAGE Standards were from Bio-Rad Laboratories (Hercules, CA, United States). AEBSF, agarose I, bovine serum albumin (BSA), CHAPS, dithiothreitol (DTT), leupeptin, mineral oil, and TG-SDS buffer concentrate were from Amresco (Solon, OH, United States). Acetic acid was from Anachemia (Montreal, Quebec); sodium dodecyl sulfate (SDS) was from J. T. Baker Chemical Co. (Phillipsburg, NJ, United States); mass spectrometry-grade trypsin was from G-Biosciences (St. Louis, MO, United States); Coomassie Brilliant Blue G-250 (CBB) was from Genlantis (San Diego, CA, United States); and Broad-range (200–10 kDa) Unstained Protein Standard was from New England Biolabs (Ipswich, MA, United States). Ammonium persulfate and aprotinin were from Thermo Fisher Scientific (Waltham, MA, United States). Acetonitrile, formic acid, and methanol were from EMD Millipore (Burlington, MA, United States). Acrylamide/bis-acrylamide (37.5:1) solution and all other chemicals utilized were from Alfa Aesar (Haverhill, MA, United States). Double glass-distilled water (ddH₂O) was used throughout.

Percoll‐washed sperm cells were sonicated and sperm heads and tails were isolated as described previously.
²³ (link) Whole sperm cells, sperm heads and tails were lysed in radio immune preciptation assay buffer (Thermo Scientific) with freshly added protease inhibitors: aprotinin, leupeptin, pepstatin, and PMSF (Thermo Scientific) on ice for 30 min followed by a 15 min spin at 14,000 x g, 4°C. Sperm lysates or CRISP2 precipitates were denatured in 4x sodium dodecyl sulfate (SDS) sample buffer (200 mM Tris‐HCl, pH 6.8, 10% β‐mercapto‐ethanol, 8% SDS, 0.08% bromophenol blue, 40% glycerol) and boiled for 10 min. Samples were loaded onto SDS‐PAGE gels (5% stacking gel, 12% resolving gel) and were blotted onto 0.2 µm nitrocellulose membranes (GE Healthcare) at 100 V for 1 h. After blocking for 3 h at RT in 5% (w/v) BSA in PBS with 0.05% (v/v) Tween‐20 (PBST), membranes were incubated with primary antibodies (Supplementary Table S1) overnight at 4°C. After three washes in PBST for 15 min, membranes were incubated with horse radish peroxidase conjugated secondary antibodies (Table S1) for 1 h at RT. After rinsing four times in PBST for 20 min, membranes were developed using chemiluminescence (enzyme chemilumenescence [ECL]‐detection kit; Supersignal West Pico, Pierce). Migration levels of proteins were visualized using a pre‐stained protein ladder, 10 to 250 kDa (Thermo Scientific).

Mycelia from GMM agar plates were collected and stored at −80°C prior to protein extraction. For protein extraction, the lysis buffer consisted of 100 mM triethylammonium bicarbonate (TEAB) with 1× Halt protease inhibitor cocktail (100×), with the final concentration of each component being 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], 800 nM aprotinin, 50 μM bestatin, 15 μM E64, 20 μM leupeptin, and 10 μM pepstatin A (Thermo Scientific, Rockford, IL) and 200 µg/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO). Mycelia were homogenized directly using Precellys 24 homogenizer (Bertin, Rockville, MD) in which each sample was processed inside a 2-ml cryotube with 0.5-mm glass beads three times (at 4°C and 6,500 rpm for 1 min., repeated 3 times with 15-s pauses in between). The lysed fungi were centrifuged at 17,000 × g for 15 min. Protein concentrations in the supernatants were measured by the Bradford assay with albumin for the standard curve (Bio-Rad Laboratories, Inc., Hercules, CA).