Fatp2

Ice-cold RIPA buffer (15 mM NaCl, 50 mM Tris HCL PH 7.4, 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate) in the presence of proteases (Roche) and phosphatases (Sigma) inhibitors cocktails was used to cell lysis. After 30 min of ice incubation, the lysates were cleared via centrifugation at 16,000 g for 15 min at 4 °C, and the protein concentration was determined using the BioRad Dc protein assay as recommended by the manufacturer (Bio-Rad, Marnes-La-Coquette, France). Proteins were mixed with 5X Laemmli loading buffer and incubated 10 min at 95 °C. Fifty μg of proteins were migrated in a 10% SDS-PAGE and transferred in a nitrocellulose membrane. The membranes were probed overnight at 4 °C with primary specific antibodies (STAT5, pSTAT5 (Cell Signaling, Danvers, MA, USA), HSC70 (Santa Cruz, Santa Cruz, CA, USA) and FATP2 (Abcam, Cambridge, UK)) after 45 min of non-specific binding sites blocking with TBS 0.1% Tween 5% nonfat milk (pSTAT5) or bovine serum albumin (STAT5, FATP2 and HSC70). The membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories). Immunoblot was revealed using Clarity Western ECL Substrate kit and ChemiDoc imaging system (Bio-Rad).

White adipose tissue was homogenized in homogenization buffer (150mM NaCl, 10mM Tris-HCl, 2mM EDTA, pH 7,4) and liver tissue in RIPA-buffer (Radioimmunoprecipitation assay buffer 150 mM NaCl, 2mM EDTA, 0,35% Na-deoxycholate, 0,50% Nonidet-40, 0,10% SDS both buffers were supplied with complete protease inhibitor (Sigma-Aldrich, Merk KGaA, Darmstadt, Germany) and phosphoSTOP (Sigma-Aldrich) using a Teflon douncer or a bead beater. Samples were incubated on ice for 30 min and further centrifuged at 10 000xg for 10 min at 4°C and the supernatants were collected.
Total protein concentration was determined using Bradford assay (BioRad). Total proteins were separated using 10 or 12% gradient SDS-polyacrylamide gel (BioRad) under denaturing conditions. Proteins were transferred onto nitrocellulose filters (Nitropure), and blots were probed with antibodies against phosphorylated and total HSL, GAPDH, ASCL1 (Cell Signaling Technology, Inc, Danvers, MA, USA) and FATP2 (abcam, Cambridge, UK), followed by detection by IRDye^TM 800 anti-Goat IgG and IRDye^TM 680 anti-Rabbit IgG antibodies using The Odyssey^® Imaging System (LICOR).