Crabp2

Western blotting was conducted using antibodies against CRABP2 (Proteintech, Chicago, IL, USA; 1:200), FABP5 (Proteintech, Chicago, IL, USA; 1:200), RAR-β (Bioss. Inc., Beijing, China; 1:150), PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200), pro-caspases-3, and active-caspases-3 (Abcam Inc., Cambridge, UK; 1:500 and 1:500). The experiment was performed by the method described elsewhere [15 (link)]. Briefly, the sample proteins (20 µg/well) were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckin ghamshire, UK). The membrane was blocked in 5% skimmed milk (Sigma-Aldrich) Tris-buffered saline (TBS-T) (10 mM Tris–HCl, pH 8.0, 0.5% Tween 20 and 150 mM NaCl) at 4 °C overnight. After three washes with TBS-T, the membrane was incubated for 3 h with the primary antibody at room temperature, followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Zymed Lab Inc., San Francisco, CA, USA). The enhanced chemiluminescence system (Roche, Penzberg, Germany) was used to detect the bound antibody. The labeling signal was removed with a stripping buffer (62.5 mM Tris–HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and the membrane was reprobed with another antibody until all the parameters were examined.

Total protein extracted from cell pellets or tissue samples were firstly quantified using the Pierce^® BCA Protein Assay Kit (Thermo Scientific) and then 80 μg protein were loaded on the SDS-PAGE gel for electrophoresis. Afterwards, the protein were transferred to the nitrocellulose membranes (NC, millipore), which were then blocked with 5% non-fat milk and incubated with the indicated primary antibodies, namely GAPDH (Proteintech, 60004-1-AP), β-actin (Sigma, A1978), Vimentin (Cell Signaling Technology, #5741), CRABP2 (Proteintech, 10225-1-AP), E-caderin (Abcam, ab76055), at 4°C overnight. The next day, the membranes were incubated with appropriate secondary antibodies conjugated with fluorescence [Licor, 926–32210 (Mouse) or 926-32211(Rabbit)]. Finally, the membranes were visualized using the Odyssey Infrared Imaging System (Licor).