FIGURE 7.

PNPLA3 has thioesterase activity. PNPLA3-WT, PNPLA3-I148M, or PNPLA3-S47A was incubated for 20 min at 37 °C with [¹⁴C]oleoyl-CoA (A), [¹⁴C]palmitoyl-CoA (B), or [¹⁴C]stearoyl-CoA (C). Lipids were extracted, applied to TLC plates, and resolved in chloroform/methanol/ammonium hydroxide (65:25:5, v/v/v). The activity of the released free oleate was analyzed by scintillation counting and calculated after normalization to the levels obtained using buffer alone. The final values obtained were plotted using GraphPad Prism 5 software. The experiments were performed three times for each substrate, and the results from one representative experiment are shown.