Fluorometric acetyltransferase activity assay kit

The enzymatic activities of ACAA1 WT and ACAA1 p.N299S were measured using the Fluorometric Acetyltransferase Activity Assay Kit (Abcam, ab204536), as previously described.^{35 (link)} Briefly, the assay was performed with 5 μg of pure protein and 100 nM acetoacetyl coenzyme A sodium salt hydrate (Sigma, A1625), and fluorescence was detected at Ex/Em of 380/520 nm. Both ACAA1 WT and ACAA1 p.N299S proteins were extracted using TnT® Quick Coupled Transcription/Translation Systems (Promega, L1170) and purified using the His-tag Protein Purification Kit (Beyotime, P2226). Each sample was analyzed in triplicate.
Lysosomal activities were determined by using the NAG assay. Briefly, lysates of HM, HM ACAA1 KO, U251-APP, or U251-APP ACAA1 KO cells treated with or without the indicated chemicals or transfected with the indicated expression vectors were isolated. The NAG assay was performed by using a commercial kit from MIBio (Cat. #SU-B16484) following the manufacturer’s instructions.

The activity of HMG-CoA reductase enzyme in the liver was assayed by the method of Rao and Ramakrishnan [42 (link)]. The enzyme activity of ACAT2 was performed using the fluorometric acetyltransferase activity assay kit (Abcam ab204536) as per the manufacturer's instructions, with fluorescence measured at Ex/Em = 380/520. The enzyme activity of LPL was performed using the fluorometric LPL activity assay kit (Roar Biomedical Inc) as per the manufacturer's instructions, with fluorescence measured at Ex/Em = 370/450. The LPL activity assay results were normalised to the amount of protein (nmol/mg of tissue or serum protein) and determined using the Bradford assay using bovine serum albumin as standard. The enzyme activity of LCAT was performed using the fluorometric LCAT activity assay kit (Roar Biomedical Inc.) as per the manufacturer's instructions, with fluorescence measured at two different wavelengths: (i) Ex/Em = 340/390 and (ii) Ex/Em = 340/470. The ratio of the two emission intensities (390 nm/470 nm) was used to express LCAT activity.