Anti protease cocktail

Kluyveromyces lactis cells harbouring the PDE5A plasmids (Fig. 2b) were grown under shaking conditions at 28 °C for up to 5 days in 5 mL of YP supplemented with glucose (YPD) or glycerol (YPG) at the concentration of 2%. Harvest cells were collected by centrifugation, washed twice with distilled water and resuspended in 300 μL of lysis buffer (20 mM Tris–HCl buffer pH 7.2, 0.2 mM EGTA, 5 mM MgCl2, 5 mM β-mercaptoethanol, 0.1% v/v Triton X-100, 2% v/v antiprotease cocktail (Sigma Aldrich, CA, USA), 1 mM PMSF) and broken with glass beads (∅ 0, 5 mm). The lysate was recovered by centrifugation at 14,000g for 30 min at 4 °C, ready for further analyses. The recovered pellets were resuspended in 200 μL of lysis buffer and analysed for PDE activity.
PDE activity was measured at 30 °C with the two-step method described by [39 (link)] using [³H]cGMP (Perkin Elmer, MA, USA). Aliquots of extracts were incubated in 60 mM HEPES pH 7.2 assay buffer containing 0.1 mM EGTA, 5 mM MgCl₂, 0.5 mg/mL bovine serum albumin, 30 µg/mL soybean trypsin inhibitor, in a final volume of 0.15 mL (PDE assay buffer). The reaction was started by adding tritiated substrate at a final concentration of 1 μM [³H]cGMP and stopped by adding 0.1 M HCl. The Sildenafil used in enzymatic inhibition experiments was a generous gift from Pfizer.

After BAL, the entire lung was perfused with isotonic solution through the right heart ventricle to flush the vascular content and lungs were weighed and frozen at −20°C until use. The right lung was homogenised in PBS containing anti‐protease cocktail (# P8340; Sigma) and centrifuged, and the supernatant was aliquoted and stored at −20°C until analysis. EPO activity was determined in lung supernatants by colorimetric assay. Following centrifugation, 100 μL of supernatants was placed in a plate with 50 μL substrate solution, corresponding to 11 mL Tris HCl + 200 mm OPD pellets + 100 μL 30% H₂O₂). After 1‐h incubation at 37°C in a shaker, EPO activity was determined as 490 nm absorbance against medium. The right lung post‐caval lobe was removed and placed in RNAlater (Thermo Fisher Scientific) for 24 h and snap‐frozen for further analysis. The left lung was removed and preserved in 4% formaldehyde for histopathological analysis.