Ge imagequant las 500

Cell fractions were isolated for RNA and protein analysis. For RNA, we used PARIS™ Kit (Thermo-Fisher Scientific) to extract nuclear and cytoplasmic RNA respectively, according to the manufacturer’s instructions. The expression level of target genes in two fractions was normalized with input RNA, which was set to 100%. For western blotting analysis, we gathered cells from culture plates and washed them twice with PBS. Then, suspend cells gently in EBC1 lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.05% NP-40, 1 mM EDTA, 1 mM DTT). Incubate cells for 5 minutes on ice and then centrifuge them 500-1000 g, 5 minutes at 4°C. The supernatant was cytoplasm, and the precipitate was washed three times with EBC1 lysis buffer, then lysed 10 minutes with NTEN lysis buffer (1 M Tris-HCl, 100 mM NaCl, 0.5% NP-40, 0.5 M EDTA) to harvest nucleus. Afterwards, protein samples were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electransferred to a nitrocellulose membrane. Next, the samples were blocked in 5% skimmed milk for 1 hour and then incubated in the corresponding primary antibody at 4°C, overnight. By the next day, they were incubated with the corresponding secondary antibody at room temperature for 1 hour. The bands were visualized by an ECL chemiluminescence detection system (GE ImageQuant LAS 500, Massachusetts, USA).

After treatment, rats in each group were used for the preparation of protein extracts. Total proteins were extracted from liver or adipose tissues samples using the radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (1 mmol/L; Sigma) and a protease and phosphatase inhibitor cocktail (100×; Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein samples were separated using 10% (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidenedifluoride (PVDF) membrane. After washing, the membranes were incubated overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal antibodies against CPT1A (1:1000; Abcam, Cambridge, UK), HADH (1:1000; Proteintech, Rosemont, IL, USA), PPARγ, Perilipin, GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA). After further washing, the membranes were incubated for 1 h with corresponding horseradish peroxidase conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG, 1:10,000; Abcam, UK). The immunoreactive bands were visualized using an enhanced chemiluminescent substrate (MILLIPORE, Burlington, MA, USA) with a GE ImageQuant LAS 500 (GE Healthcare, USA). The intensity of the protein bands was quantitated using a Gel Doc XR system (Bio-Rad, Hercules, CA, USA).