Western Blot Analysis of 3T3-L1 Cell Lysates

WB analysis was performed as described in [28 (link)]. 3T3-L1 cell lysates were obtained by lysing cells in buffer containing 20 mm Tris-HCl, pH 7.5; 5 mm Ethylenediaminetetraacetic acid (EDTA); 150 mm NaCl; 1% Nonidet P40 (NP40), 10 μm phenylmethylsulfonyl fluoride (PMSF); 5 μg/mL aprotinin; and 5 μg/mL leupeptin. Protein concentration was determined by Coomassie blue protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins lysates were then analyzed by SDS-PAGE, and then electrophoretically transferred to a Polyvinylidene fluoride (PVDF) membrane. Membranes were probed with antibodies to total CREB (48H2, #9197; Cell Signaling Technology, Danvers, MA, USA), C/EBPβ (C-19, sc-150; Santa Cruz Biotechnology, Dallas, TX, USA), and VINCULIN (7F9, sc-73614; Santa Cruz Biotechnology, Dallas, TX, USA), and successively re-probed with secondary mouse or rabbit antibodies (Bio-Rad Laboratories) before signal detection with Enhanced chemiluminescence (ECL) plus (GE Healthcare, Chicago, IL, USA).