Frozen tissues (0.5 100 mg) were homogenized using a Physcotron homogenizer in QIAzol Lysis Reagent (Qiagen) and total RNA was extracted using an RNeasy Mini Kit (Qiagen).
For Nampt/PBEF/visfatin analysis on mRNA level, tissue samples (30 40 mg) were homogenized using Fastprep 24 Homogenizer (MP Biomedical, USA).
Similarly, muscle tissues from Patients 2 and 4 and two control subjects (∼30 mg) were homogenized using a Teflon glass Dounce homogenizer at 4°C (20 strokes) in homogenization buffer containing 250 mM sucrose, 20 mM Imidazole-HCl pH 7.4 and 1 mM PMSF.
Frozen muscle tissues of 10 30 mg were homogenized in H2O by Shakeman homogenizer (BMS, Tokyo, Japan) using 100 μl H2O for every 10 mg of tissue.
Samples (60 100 mg) were homogenized using a Potter-Elvehjem homogenizer on ice in 10 volumes of extraction buffer (Tris 50 mM, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% DOC, 1% NP-40, 1% SDS, 1 mM DTT, protease inhibitors from Roche, 1mM PMSF), and an equal amount of protein (6 µg per well) was loaded and run on 10% SDS-PAGE.
Frozen liver samples (~300 mg) were homogenized on ice with an Ultraturrax homogenizer (IMLAB Sarl, Lille, France).
Subsequently, tissue samples weighing 50 ± 10 mg were homogenized in a total volume of 1 mL TRI Reagent using a homogenizer, and total RNA was isolated according to manufacturer instructions (Sigma, Sintra, Portugal).
Samples (200 mg) were homogenized in G2 Lysis Buffer containing 80 μg/ml RNaseA, using a hand-held homogenizer (Polytron, Switzerland), and thereafter digested with 1 mg/ml Proteinase K (Roche Diagnostics, Burgess Hill, UK) overnight.
Frozen brain samples (midbrain, ~50 mg) were homogenized in methanol (1 ml) containing [2H4]-anandamide, [2H4]-PEA, [2H4]-OEA, and [2H8]-2-AG as internal standards.
Tissues (0.1 mg) were homogenized in 0.5 mL 1 × RIPA lysis buffer (EMD Millipore, Billerica, MA) supplemented with protease inhibitor cocktail (Sigma-Aldrich).
To asses FAAH activity, tissue samples from brain (cerebellum, ~100 mg) and liver (~100 mg) were homogenized in 10 volumes of ice-cold Tris-HCl buffer (50 mM, 5 9 vol, pH 7.5) containing 0.32 M sucrose and centrifuged at 1000 g for 10 min at 4 °C.
