Halt

A piece of WAT or heart was homogenized with a polytron disperser (IKA T10 basic ULTRA-TURRAX, Königswinter, Germany) in a proportion 1:10 in STE buffer (250 mM sucrose, 20 mM Tris-HCl, 40 mM KCl, 2 mM EDTA, pH 7.4). Homogenates were stored at −20 °C with protease and phosphatase inhibitors (HALTTM; Thermofisher Scientific, Waltham, MA, USA) until Western blot analysis. Protein concentration was determined by BCA method (Thermofisher Scientific, Waltham, MA, USA). Heart triglycerides were determined in fresh homogenate (STE buffer without proteases and phosphatases inhibitors) with a commercial kit according to manufacturer indications (Cromatest, Barcelona, Spain; Wako Chemicals GmbH, Neuss, Germany). Total lipid content of WAT was quantified by Folch method [64 (link)].

Exponentially growing cells were incubated in isotonic or hypotonic medium as indicated. Cells were collected by trypsinization (without EDTA) and lysed in lysis buffer (50 mM Tris-HCl pH 8, 5 mM CaCl₂, protease inhibitors (1:100, Halt^TM, Thermo Fisher Scientific, Waltham, MA, USA), 1% IGEPAL CA-630 (Sigma-Aldrich^®, Merck KGaA, Darmstadt, Germany) (50 µL per 0.5 × 10⁶ cells) for 30 min on ice. Digestion was performed with 10 U MNase solution (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 37 °C and stopped by adding an equal volume of stop buffer (400 mM NaCl, 40 mM EGTA (Sigma-Aldrich^®, Merck KGaA, Darmstadt, Germany), 2% SDS (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 2.5% Proteinase K (Macherey-Nagel, NucleoSpin Tissue Kit). Samples were purified using Macherey-Nagel NucleoSpin Gel and PCR clean-up kit according to manufacturer’s instructions. Entire eluate was run in a 1% agarose gel cast with ethidium bromide (EtBr), in TAE buffer (40 mM Tris-HCl, 20 mM acetic acid, 1 mM EDTA) at 70 V for 2 h. Gels were scanned and digitized on a Typhoon Imager (GE Healthcare, Buckinghamshire, UK).