Ecl system reagents

Samples containing equal amounts of total protein were mixed with gel loading buffer (50 mM Tris, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/mL bromophenol blue) in a ratio of 4:1 (v/v) and incubated at 95 °C for 5 min. Samples (30 μg of protein) were separated on SDS-polyacrylamide gels (12%) (Mini Protean II, Bio Rad, Hercules, CA, USA) using the Laemmli buffer system and proteins were semidry transferred to nitrocelulose membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked for 1 h with 5% (w/v) nonfat dried milk in TTBS and incubated overnight at 4 °C with specific primary antibodies: anti-SCD1 (1:500, R&D Systems, Minneapolis, MN, USA), anti-SREBP1 (1:1000, Novusbio, Littleton, CO, USA), anti-β-ACTIN (1:5000, Sigma, Saint-Louis, MO, USA) then for 1 h with HRP-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA). Bands were developed with the use of ECL-system reagents (GE Healthcare, Chicago, IL, USA). Rainbow markers (Amersham Biosciences, Amersham, UK) were used for molecular weight determinations. Protein pattern images were taken using an ImageQuant Las 500 (GE Healthcare, Chicago, IL, USA). Data analysis was performed using Image Lite Studio software (LI-COR, Lincoln, NE, USA).

Immunoblotting analysis used to determine the expression of CSAD was conducted as previously described [50 (link)]. Briefly, the samples were separated on SDS-polyacrylamide gels (7.5–15%) (Mini Protean II, Bio-Rad, Hercules, CA, USA) using the Laemmli buffer system and semidry transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked overnight at 4 °C with 5% (w/v) non-fat dried milk in TTBS and incubated for 3 h at room temperature with specific primary antibodies followed by 1 h incubation with HRP-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA). Bands were developed with the use of ECL-system reagents (GE Healthcare, Chicago, IL, USA). GAPDH was used as a control of equal protein content. The following specific primary antibodies were applied: ANTI-CSAD (MyBioSource, San Diego, CA, USA) and ANTI-GAPDH (MyBioSource, San Diego, CA, USA). The images were visualized using the ImageQuant Las 500 (GE Healthcare, Chicago, IL, USA) and analyzed by Image Lite Studio software (LI-COR, Lincoln, NE, USA).