Anti alox12

Samples were separated by electrophoresis in 4 to 20% SDS–polyacrylamide gel electrophoresis gels with 20 mg per lane, transferred to a polyvinylidene difluoride membrane, and blotted with antibodies. Secondary horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) and Amersham ECL Prime Western Blotting Detection Reagents (catalog no. RPN2232, GE Healthcare) were used for detection. Antibodies used during immunoblotting include anti-actin (Santa Cruz, sc-1616), anti-RARγ (Santa Cruz, sc-7387), and anti-Alox12 (Santa Cruz, sc-365194).

Proteins were electrophoresed by SDS/PAGE (12% or 10% gel) and the blots were incubated overnight with primary antibody. The following primary antibodies were used: anti-ALOX12 (Santa-Cruz, sc-365,194), anti-E-Cadherin (Cell signal Technology, #14472), anti-N-Cadherin (Cell signal Technology, #13116), anti-Vimentin (Cell signal Technology, #5741), anti-Snail (Cell signal Technology, #3879), anti-Slug (Cell signal Technology, #9585), anti-MMP2 (Abcam, ab37150), anti-MMP7 (Abcam, ab5706), anti-MMP9 (Abcam, ab38898), anti-MMP13 (Abcam, ab39012), anti-GPR31 (Abcam, ab75579), anti-PI3K (Cell signal Technology, #4249), anti-AKT (Abcam, ab179463), anti-AKT (phospho T308, Abcam, ab38449), anti-NF-κB (Abcam, ab16502), anti-NF-κB (phospho S536, Abcam, ab86299), anti-GAPDH (Abcam, ab181602).

Cells and frozen liver sections (4 μm) fixed with acetone were penetrated with 0.3% Triton for 15 min. Then the sections were block with 10% fetal sheep serum, followed by incubation with primary antibodies overnight at 4 °C. After washing, sections or cells were incubated with corresponding secondary antibodies, followed by incubation with DAPI. After mounting, the slides were observed under immunofluorescence microscope. The following antibodies were used: anti-ALOX12 (Santa-Cruz, sc-365,194), anti-Vimentin (Cell signal Technology, #5741), anti-MMP9 (Abcam, ab38898), Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (Abcam, ab150117).