Leupeptin

Reagents used for protein expression and purification include: 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, Fisher BioReagents 30827-99-7), phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich P7626), Tosyl-L-lysyl-chloromethane hydrochloride (TLCK, Sigma-Aldrich T7254), leupeptin (Thermo Scientific 78435), benzamidine (Sigma-Aldrich B6506), FLAG affinity resin (Sigma-Aldrich A2220), FLAG peptide (Sigma-Aldrich, F3290) and HIS-Select resin (Sigma-Aldrich P6611), and biotin (Sigma-Aldrich B4639).

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).

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).

Reporter-cells were washed briefly with ice-cold DPBS and lysed with 50 µl of lysis buffer per well (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% IgePAL, 0.5% Sodium deoxycholate and freshly added 250 µM PMSF, 10 ng Leupeptin, 10 ng Aprotinin, 1 ng Pepstatin A, 10 ng DNase und 1 µl Halt Protease Inhibitor Cocktail; Thermo Fisher Scientific). Cell lysates were collected into a pre-cooled tube and were centrifuged for at least 1 h at ~15,000 × g at 4 °C. The supernatant was collected into two tubes, snap-frozen with liquid nitrogen and stored at −80 °C until needed.