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. 2014 Oct 7;9(10):e109869. doi: 10.1371/journal.pone.0109869

Figure 4. Fatty acid liberation from various substrates when incubated together with lysates of HEK293 cells expressing hBAT5 or hABHD12.

Figure 4

HEK293 cells were transiently transfected with the cDNA encoding hBAT5 or hABHD12, as detailed in [10] and the Methods section. After 48 h, cells were harvested and lysates prepared for lipase activity measurements based on the Cayman’s FFA fluorescence assay kit. For validation purposes, lysates of parental HEK293 cells were tested in parallel, and the substrate panel included the MAGs 1-AG (A) and 1-LG (B), the lysophospholipids C18∶1-LPA (C) and C18∶1-LPS (D), the DAG 1,2-dioleoyl(C18∶1)-rac-glycerol (E), and the TAG 1,2,3-trioleoyl(C18∶1)glycerol (F). Cellular lysates (0.3 µg/well) were incubated together with the indicated substrates [25 µM final concentration, added from 10 mM stock solutions in ethanol into the FFA assay mix containing 0.1% (v/v ethanol). Assay blank and fatty acid standard (C18∶1) were included for each condition. FFA content was determined at time-points 10 and 20 min. Data are mean ± SEM from quadruplicate wells in one experiment. Statistical comparisons to values of the HEK lysates were done using paired t-test and the significance is indicated with an asterix (NS, non-significant; **, p<0.01; ***, p<0.001).