Lipid analysis
Sterol and fatty acids were determined as described by Chalbi et al. , (2015). A mixture of chloroform-methanol (1:2, 0.75 mL) was added in an Eppendorf tube to both, microsomal and plasma membrane fractions (0.5 mL), along with β-cholestanol (20 µL, 0.1 mg mL-1) used as internal standard for sterol analysis. Chloroform (CHCl3; 0.25 mL) was added and the mixture was shaken and centrifuged at 10,000 x g for 6 min. The CHCl3 layer was retained, evaporated to dryness under N2 and made up to 100 µL with CHCl3. For sterol analysis, 20 µL of the CHCl3 extract was placed in a glass vial (2 mL), evaporated to dryness under N2and acetylated using pyridine (50 µL) and Ac2O (100 µL). After 2h, the solvents were evaporated under N2, ethyl acetate was added and the sterols analyzed by GC using an HP5-bonded capillary column (30 m x 0.25 mm x 0.25 µm) coupled to a flame ionization detector (FID), with H2 as carrier (1mL min-1) and a temperature programmed of 120-260°C at 5°C min-1, then 260-280°C at 2°C min-1, and finally 280-300°C at 6°C min-1. The injector and detector temperatures were 150 and 320°C, respectively. Bound fatty acids were determined by using 20-II portions of the CHCl3 extract; evaporating them to dryness under N2, transmethylating with sodium methoxide (0.5 N) in methanol (0.5 mL) and heating at 30°C for 7 min. The resultant fatty acids methyl esters were extracted with hexane (1 mL), evaporated under N2, dissolved in ethyl acetate (20 µL) and analyzed by GC using an HP5-bonded capillary column (30 m x 0.25 mm x 0.25 µm), with FID, He as carrier (1 mL min-1) and a temperature programmed of 150-195°C at 3°C min-1, then 195-220°C at 2°C min-1, and finally 220-300°C at 6°C min-1. The injector and detector temperatures were 280 and 300°C, respectively.