Journal: Journal of Lipid Research
Article Title: Arylacetamide deacetylase attenuates fatty-acid-induced triacylglycerol accumulation in rat hepatoma cells
Figure Lengend Snippet: AADA hydrolyzes soluble and insoluble carboxylesters. Twenty micrograms of membrane protein prepared from McA cells stably transfected with an empty pCIneo vector, FLAG-tagged mouse AADA, or human TGH cDNAs were incubated with pNP-acetate (A) or 4-MUH (B). The release of pNP was monitored at 405 nm and that of 4-umbelliferyl at 460 nm. The results are presented as an average from triplicate measurements. C: Hydrolysis of microsomal lipids. Cells were incubated with radiolabeled oleic acid, and microsomes were isolated and incubated for 4 h as described in Materials and Methods. Lipids were extracted and resolved on TLC plates, and radioactivity was determined by scintillation counting. Data (mean ± SD) from three separate experiments performed in triplicate are presented with radioactivity in DG in pNeo microsomes at time 0 set as 100%. Equal amounts of microsomal protein were used for the assay. The actual starting dpm in microsomal lipid fractions at time 0 were 22,000–25,000 for PC, 12,000–18,000 for TG, and 1,500–2,300 for DG. D: Thirty micrograms of total cellular membrane proteins from McA cells stably transfected with pCI-neo, A13, A23, and human TGH were either not treated (pNeo-NT) or treated with FP-biotin, resolved in SDS-PAGE, and transferred to nitrocellulose membrane, and biotinylated proteins were visualized with streptavidin-HRP. Asterisks indicate other potential endogenous serine hydrolases present in McA cells.
Article Snippet: Following transfer to nitrocellulose, biotinylated proteins were visualized by probing with avidin-HRP and enhanced chemiluminescence (ECL).
Techniques: Stable Transfection, Transfection, Plasmid Preparation, Incubation, Isolation, Thin Layer Chromatography, Radioactivity, SDS Page