FIGURE 9.

Ceramide synthase activity of the FAM57B protein. A, in vitro ceramide synthase activity was measured using differentiated ST2 cell extracts (50 μg of protein), which overexpressed each FAM57B variant (var1–var3) with different substrates, such as palmitoyl-CoA (C16:0), stearoyl-CoA (C18:0), arachidoyl-CoA (C20:0), and behenoyl-CoA (C22:0). B, an in vitro ceramide synthase assay was performed using undifferentiated FAM57B-FLAG or ceramide synthase 2-FLAG (CERS2F)-overexpressed ST2 cell extracts (50 μg of protein) that were preincubated with or without 20 μm FB1, followed by adding C18-acyl-CoA as substrate. C, in vitro ceramide synthase activity with or without fumonisin B1 was assayed using 293FT cell extracts (50 μg of protein), which transiently overexpressed FAM57B var2F or CERS2F. D, ST2 cells overexpressing CERS2F, EGFP, or FAM57B-F (each variant) were immunoprecipitated (IP) with an anti-FLAG antibody, which was released with 3× FLAG peptide, and subjected to Western blot analysis (WB) using an anti-FLAG antibody. E, in vitro ceramide synthase activity was measured using purified FAM57B-FLAG protein (var1F–var3F) and CERS2F, with different substrates, such as C16-, C18- and C20-acyl-CoA. Results are representative of three independent experiments.