Western Blotting of CRABP2, FABP5, RAR-β, PPAR-β/δ

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.