Dmem medium

AC29 cells, an ACAT1-deficent Chinese hamster ovary (CHO) mutant cell line [28 (link), 29 (link)], were maintained in 50% F-12 medium (Corning, Manassas, VA, USA) and 50% DMEM medium supplemented with 10% calf serum (Atlanta Biologicals, Flowery Branch, GA, USA). HEK293 cells were maintained in DMEM medium supplemented with 10% newborn calf serum (Atlanta Biologicals, Flowery Branch, GA, USA). All cell lines were maintained at 37°C under humidified conditions and 5% CO₂.
The expression plasmid HisACAT1/FLAG was constructed as described previously [30 (link)]. In brief, an octapeptide FLAG-tag was inserted at the C-terminus of human ACAT1 cDNA containing a 6-histidine (His)-tag at the N-terminus. This fragment (HisACAT1/FLAG) was ligated into the mammalian expression vector pAG3-Zeo [31 (link)]. The expression plasmid Δ1-65 HisACAT1/FLAG was constructed in a similar manner, by using the HisACAT1 cDNA devoid of the first 65 amino acids [32 (link)]. HisACAT1/FLAG was purified to homogeneity as previously described [12 (link), 30 (link)] from stably expressing HEK293 cells by promptly purifying the enzyme first with HisPur Ni-NTA resin and subsequently with anti-FLAG M2 resin. AC29 cells stably expressing HisACAT1/FLAG or Δ1-65 HisACAT1/FLAG were isolated as previously described [12 (link)].

All cell lines were maintained at 37°C under humidified conditions and 5% CO₂. HEK293 cells were maintained in DMEM medium (Corning, Manassas, VA, USA) supplemented with 10% newborn calf serum (Atlanta Biologicals, Flowery Branch, GA, USA). CHO cells were maintained in 50% F-12 medium (Corning, Manassas, VA, USA) and 50% DMEM medium supplemented with 10% calf serum (Atlanta Biologicals, Flowery Branch, GA, USA).