CERS3 regulates activities of ELOVL1 and ELOVL4 in differentiated keratinocyte.

<p>(A–C) Keratinocytes were transfected with control or <i>CERS3</i> siRNA. Twenty four h after transfection, transfection media were changed to differentiation medium, and the cells were incubated for an additional 2 days. (A) Total RNA was prepared from the cells, then subjected to real-time PCR using primers specific for <i>CERS3</i> and <i>GUSB</i> as an internal control. The expression level of <i>CERS3</i> mRNA was calculated by normalizing to that of <i>GUSB</i>. Values presented are the amount of <i>CERS3</i> mRNA relative to that in control cells and represent the mean ± S.D. from three independent experiments. Statistically significant differences to control siRNA are indicated (*p<0.05; the Student’s t-test). (B) Total cell lysates (10 µg protein) prepared from the cells were subjected to immunoblotting with an anti-CERS3 antibody or anti-CERS2 antibody, or with an anti-GAPDH antibody to demonstrate uniform protein loading. (C) Total membrane proteins (40 µg) were prepared and subjected to an <i>in vitro</i> FA elongase assay by incubating with 50 µM C22∶0-CoA or C26∶0-CoA and 0.075 µCi [¹⁴C]malonyl-CoA, for 30 min at 37°C. After termination of the reactions, lipids were subjected to methanolysis, extraction, separation by reverse-phase TLC, and detection by an FLA7000 bioimaging analyzer. Values indicate the radioactivities of the FA methyl ester products relative to that of control siRNA-transfected cells, and represent the mean ± S.D. of three independent experiments. Statistically significant differences to control siRNA are indicated (*p<0.05, **p<0.01; Student’s t-test).