Western Blot Analysis of Phosphorylated CREB

WB analysis were performed as previously described [46 (link)]. Briefly, 3T3-L1 cells were harvested and washed in PBS 1X, and then lysed in Lysis Buffer containing 20 mM Tris-HCl, pH7.5, 150 mM NaCl, 5 mM EDTA, 1% NP40, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 10 μM PMSF. Samples were incubated on ice 30 min after the addition of Lysis Buffer and then cell lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C. The protein concentration of the cell lysate was determined using the Coomassie blue protein assay (Bio-Rad Laboratories, Hercules, CA). Protein lysates were then analyzed by SDS-PAGE, transferred to a PVDF membrane and subjected to WB analysis. Membranes were firstly probed with antibodies to phospho-CREB Ser133 (1B6; #9196) and CREB (48H2; #9197) from Cell Signaling Technology (Danvers, MA) and to β-Actin (I-19; sc-1616) from Santa Cruz Biotechnology Inc (Dallas, TX) and then probed with secondary mouse or rabbit antibodies (Bio-Rad Laboratories) before detection of the signal with ECL plus (GE Healthcare, Chicago, IL).