Rabbit anti fasn c20g5

Total cell lysate was prepared with RIPA buffer with protease inhibitors (Protease Inhibitor Cocktail Tablets, Roche). Nuclear and cytoplasmic protein fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Protein samples were separated on the SDS-PAGE gels using 4-15% Mini-PROTEAN TGX Precast Gels (Bio-Rad) and then transferred to polyvinyl difluoride (PVDF) membranes (Immobilon-P, Millipore) for immunoblotting analysis. The following primary antibodies were used: mouse anti-SREBP1 (IgG-2A4, BD Biosciences), rabbit anti-FASN (C20G5, Cell Signaling), rabbit anti-SCD (23393-1-AP, Proteintech), rabbit anti-ACSL1 (D2H5, Cell Signaling), rabbit anti-ACSS2 (D19C6, Cell Signaling), rabbit anti-histone H3 (9715, Cell Signaling) and rabbit anti-actin (13E5, Cell Signaling). After being incubated with primary antibodies overnight in PBST solution with 5% non-fat dry milk, membranes were probed with HRP-conjugated affinity-purified donkey anti-mouse or anti-rabbit IgG (GE Healthcare) and visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore).

HT-144 cells were seeded in 10 cm² plates in 10% FBS/RPMI-1640 medium at day one. Sixteen hours after seeding, cells were washed twice with PBS buffer and then cultured in different medium conditions. Total cell lysate was harvested with RIPA buffer containing protease inhibitors (protease inhibitor cocktail tablets, Roche) on the third day. Nuclear and cytoplasmic protein fractions were prepared with NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Protein samples were separated on the 4–15% Mini-PROTEAN TGX precast SDS-PAGE gels (Bio-Rad) and then transferred to polyvinyl difluoride (PVDF) membranes (Immobilon-P, Millipore) for immunoblot analysis. The following primary antibodies were used: mouse anti-SREBP1 (IgG-2A4, BD Biosciences), rabbit anti-FASN (C20G5, Cell Signaling), rabbit anti-SCD (23393-1-AP, Proteintech), rabbit anti-ACSL1 (D2H5, Cell Signaling), rabbit anti-histone H3 (9715, Cell Signaling) and rabbit anti-beta-actin (13E5, Cell Signaling). After being incubated with primary antibodies overnight in PBST solution with 5% non-fat dry milk, immunoblot membranes were probed with HRP-conjugated affinity-purified donkey anti-mouse or anti-rabbit IgG (GE Healthcare) as secondary antibodies and visualized with the immobilon Western Chemiluminescent HRP substrate (Millipore).