Easypack protease inhibitors

Tissue samples (50 mg) were solubilized in Ripa Buffer (89900, Thermo Fisher, Waltham, MA, USA) with addition of EASYpack protease inhibitors (5892970001, Roche). Protein concentration in supernatants after 14,000 rpm centrifugation was determined using the BCA method. 25 mkg of protein were separated on 8% SDS-PAGE with subsequent blotting on nitrocellulose membrane (BIO-RAD) using Trans-Blot^® Turbo™ Transfer System (Bio-Rad) with subsequent immersion in 5% skimmed milk in TBST buffer.
Primary ab: 1:1000-FSTL1, (BAF1738, R&D System); GAPDH (32233, Santa-Cruz). Secondary ab: 1:10000-anti-Mouse IgG (H+L) Secondary Antibody (115-005-003, Jackson ImmunoResearch), anti-Rabbit IgG (H+L) Secondary Antibody (111-035-144, Jackson ImmunoResearch). Signal was revealed by the ECL method following the HRP reaction.

Mycelial proteins were extracted using a modified trichloroacetic acid (TCA)/acetone protocol [38 ]. Briefly, the frozen mycelia (in liquid nitrogen) were ground using a pestle and mortar and homogenised in extraction buffer: 50 mM of Tris-HCl at pH 7.5, 200 mM NaCl, 5 mM EDTA, 0.5% Triton X-100 and EDTA-free EASYpack protease inhibitors (Roche, Switzerland). To facilitate homogenisation and cell rupture, a TissueLyzer LT Adapter (Qiagen, Germany) was used, first 1 g of glass beads (half of each size: 0.5 and 0.1 mm) were added and then two consecutive cycles of 5 min at top speed were applied. Proteins were precipitated in acetone containing 10% (v/v) trichloroacetic acid (TCA) and 40 mM of dithiothreitol (DTT) (1:10 w/v) for 1 h at − 20 °C; the pellet recovered by centrifugation at 10,400 g for 15 min, washed three times in 10 mL of acetone containing 40 mM of DTT, finally dried under a nitrogen flow and stored at − 80 °C until further analysis.