Ab54787

The AML12 cells were seeded at a concentration of 3×106 cells/ml into 60 mm dishes and, once confluent, were lysed in ice-cold lysis buffer containing 1% NP-40, 50 mM Tris-HCl, 0.1% SDS, 1% sodium deoxycholate and 150 mM NaCl (pH 7.4; Tianjin Chemical Reagent Factory, Tianjin, China) and centrifuged at 12,000 × g at 4°C for 3 min. The protein content was determined using Quick Start™ Bradford kit (5000202EDU; Bio-Rad Laboratories, Inc., Hercules, CA, USA), using bovine serum albumin (BSA) as the standard. Subsequently, 15 µl of the proteins were separated by SDS-PAGE on 12% acrylamide gels, following which proteins were transferred onto a PVDF membrane (EMD Millipore, Bethesda, MA, USA). The membrane was then incubated overnight in a blocking buffer containing appropriate diluent (ab64211; Abcam, Cambridge, UK) of primary antibodies against gp78 (ab54787; 1:1,000, Abcam), cidec (ab77115; 1:1,000, Abcam), PPAR-γ (ab41928; 1:1,000, Abcam) and β-actin (ab8226; 1:1,000, Abcam) at 4°C. The proteins were detected by incubation with horseradish peroxidase-conjugated secondary antibody (ab6789; 1:2,000, Abcam) in diluent (ab64211; Abcam, Cambridge, UK) at room temperature for 1 h. Following extensive washing with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the bands were visualized using enhanced chemiluminescence and autoradiography.

The AML12 cells were seeded onto sterile coverslips and, following incubation at 37°C for 24 h, the cells were transfected with pCMV-Myc-CIDE-3 and pCMV5-HA-gp78 for 48 h, and in 5% BSA for 30 min. The cells were then incubated with primary antibodies specific for gp78 (ab54787; dilutions 1:1,000, Abcam) or cidec (ab213693; dilutions 1:1,000, Abcam) overnight at 4°C, followed by incubation with secondary antibodies conjugated with fluorescein isothiocyanate for gp78 (ab6785; dilutions 1:3,000, Abcam) and with tetraethyl rhodamine isothiocyanate for cidec (ab6718; 1:3,000, Abcam) for 1 h at 37°C. Prior to imaging, the cells were counterstained with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 37°C to stain the nuclei, and were visualized with an E1000 digital camera (Nikon Corporation, Tokyo, Japan) with SimplePCI software version 65 (Meyer Instruments, Houston, TX, USA).