Cortical tissue surrounding the subdural electrode (Fig. 5A) was sampled and homogenized in 2% SDS with protease inhibitor mixture, β-glycerophosphate (10 mM), and orthovanadate (1 mM).
The mouse brain tissues were homogenized in lysis buffer (10 mM Tris-HCL, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-β-glycerophosphate, Phosphate inhibitor mixture I and II (Sigma), and complete protease inhibitor mixture (Roche)], using a Diax 900 homogenizer (Sigma).
Subsequently, cells were rinsed twice with ice-cold PBS and solubilized in lysis buffer (10% glycerol, 1.0% Triton-X-100, 150 mM NaCl, 5 mM EDTA, 50 mM HEPES; pH 7.4) supplemented with Complete Mini protease inhibitor mixture (#11697498001; Sigma).
Briefly, the striata were homogenized in ice-cold buffer (in mM): 320 sucrose, 5 Hepes-NaOH, pH 7.4, 0.5 EDTA, 0.1 phenylmethylsulphonyl fluoride (PMSF), 1 Na3VO4, 10 NaF, and protease inhibitor mixture (Complete, Roche) using a Teflon-glass grinder.
The corrosion of GW103 in the ethylene glycol solution can be effectively inhibited by 1000 ppm of the inorganic organic inhibitor mixture.
After wash with cold PBS, ice-cold cell lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, protease inhibitor mixture (Sigma-Aldrich) and PMSF) were added to the cells for 30 min.
After measuring the weights of body and fat tissues (inguinal WAT, IgWAT; epididymal WAT, EpiWAT; interscapular BAT), IgWAT were removed and homogenized in radioimmuno-precipitation assay buffer (containing protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, USA) for further immunoblot analyses.
We added 400 μl of polysome buffer (50 mM Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/ml RNasin, 1 mg/ml heparin, 100 μg/ml cyclohexamide, and 1× protease inhibitor mixture) to each sample and disrupted the tissue in 1.5 ml microcentrifuge tubes using pellet pestles (749540-0000; Kimble Chase).
Brain samples were mechanically homogenized in RIPA buffer (50 mmol/l Tris HCl, pH 7.4/150 mmol/l NaCl/1% Nonidet P-40/0.5 P-40/0.5deoxycholate/0.1% sodiumigma) plus protease inhibitor mixture (Roche 1873580), andeoxycholate/0.1tration was measured using a BCA protein assay kit (Thermo Scientific).
The cells were centrifuged (SIGMA 3K30, Germany) for 5 min at 14,000 rpm at 4°C, and 1.5 μl of protease inhibitor (Protease Inhibitor Mixture Set I, Calbiochem) was added.
15 µg total cell lysates were prepared by briefly sonicating cell pellets in 1× lysis buffer (Cell Signaling) supplemented with a protease inhibitor mixture (Roche) and phosphatase inhibitors (PhosSTOP, Roche).
