Free protease inhibitor cocktail tablet

Cultured FLS and bone marrow-derived macrophages were lysed using a radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing cOmplete Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany). Protein concentrations of cell lysates were measured with a bicinchoninic acid protein quantification kit (Merck, Kenilworth, NJ, USA), and an equal amount of protein was separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidase-conjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

For RNA isolation, myoblasts and myotubes were collected in 500 μL Tri-Reagent (Sigma-Aldrich) and stored at −20°C until processing. For protein isolation, cells were collected in 200 μL of RIPA buffer (Millipore) containing PhosStop phosphatase inhibitor cocktail tablets (Roche) and cOmplete Mini, EDTA-free protease inhibitor cocktail tablets (Roche) on ice. Samples were then incubated on ice for 30 min with vortexing to ensure lysis and then centrifuged for 10 min at 16,000 x g at 4°C. Supernatants were collected and stored at −20°C.
Mice were euthanized and muscles were dissected and snap-frozen in liquid nitrogen and stored at −80°C. Tissue was lyophilized for 8 h and pulverized with ceramic beads on dry ice using a Bertin Technologies Percellys Evolution Tissue Homogenizer (Molnar et al., 2021 (link)). Pulverized tissues were then homogenized on ice with a Polytron PT1200-E in either 500 μL Tri-Reagent or 100 μL RIPA buffer supplemented with protease and phosphatase inhibitor cocktail tablets (Roche). Homogenized tissues in Tri-Reagent were stored at −20°C. Homogenized tissues in RIPA buffer were centrifuged for 10 min at 16,000 x g at 4°C, and the supernatant was stored at −20°C.

Kidney cortex samples were homogenized in a buffer consisting of 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 0.025 mM ZnCl₂, and 0.1 mM Pefabloc SC Plus (Roche) and EDTA-free protease inhibitor cocktail tablet (Roche) and clarified by centrifugation at 14,000×g for 10 min at 4°C. After measuring protein concentration by Micro BCA assay kit (Thermo Scientific), tissue samples were diluted in a buffer containing 100 mM Tris-HCl, 600 mM NaCl, 10 µM ZnCl2, pH 7.5 and 100 µM captopril, 5 µM amastatin, 5 µM bestatin (all from Sigma-Aldrich), and 10 µM Z-Pro-prolinal (Enzo Life Sciences). To each well, 40 µl of a diluted tissue sample (0.5 µg of total renal protein) was added, along with 10 µl of buffer (with or without an specific ACE2 inhibitor, MLN-4760), and the reaction was initiated by the addition of 50 µl of the substrate (5 µmol/l, final concentration). Kidney cortex ACE2 activity was determined after 4-hour incubation at 37°C. The plates were read as described above. Experiments were carried out in duplicate for each data point. Results after subtraction of the inhibition value were expressed as RFU (Relative Fluorescent Units) per µg of protein and per hour (RFU/µg/hr).

WT and PATL2‐HA‐tagged mice were sacrificed by cervical dislocation, and the hypothalamus, pituitary glands and livers were collected, snap‐frozen in liquid nitrogen and stored at −80°C. The day of the experiment, organs were thawed in RIPA lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, Complete EDTA‐free protease inhibitor cocktail tablet (Roche)) and homogenised using a Dounce tissue grinder before mixing the supernatant with loading buffer in equal volumes (4% SDS, 62.5 mM Tris pH 6.8, 0.1% bromophenol blue, 15% glycerol, 5% bromophenol blue). The cytoplasmic fraction was isolated by centrifugation at 3,000 × g for 10 min. GV oocytes were collected in M2 medium supplemented with 150 mg/ml dbcAMP and prepared as described above. Oocytes were washed three times in PBS‐PVP 0.5% to remove proteins from the M2 medium and added to an equal volume of loading buffer for a final volume of 20 μL. All protein samples were heated to 65°C for 15 min before loading onto a 4–20% TGX Mini‐PROTEAN stain‐free precast gel (Bio‐Rad). Proteins were transferred onto a PVDF membrane, blocked in 5% milk and incubated overnight at 4°C in anti‐HA antibody (Appendix Table S2) in blocking solution.

Muscle extracts were prepared by homogenising muscles in ice-cold buffer [0.15% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate (Na₃VO₄), 10 mM sodium fluoride (NaF), 15 mM sodium chloride (NaCl), 20 mM HEPES, 10 mM PMSF, pH 7.5] supplemented with 10 mM PMSF and 1 complete™, EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, Sydney, Australia) using a Pro200 homogeniser (PRO Scientific Inc, Sydney, Australia). Samples were incubated on ice for 1 hr and supernatant lysates were collected following centrifugation at 14,000g for 10 min at 4°C. Protein samples were divided into aliquots and kept at –80°C before subsequent experimental procedures.