Fig. 4.
CERS2 regulates ELOVL1 activity. (A) Total membrane proteins (40 μg) prepared from HEK 293T cells were incubated with the indicated acyl-CoA (40 μM) and 0.025 μCi [14C]malonyl-CoA in the presence or absence of 20 μM FB1 for 1 h at 37 °C. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by TLC, followed by detection and quantification by a bioimaging analyzer BAS-2500. Values represent the mean ± SD from three independent experiments. Statistically significant differences are indicated (*p < 0.05, **p < 0.01; t test). (B) HeLa cells were transfected with the indicated siRNAs (6 nM) 4 d prior to experiments. Total RNA was prepared then subjected to RT-PCR using primers specific for CERS2, CERS4, CERS5, or GAPDH. (C) Total membrane proteins (40 μg) prepared from HeLa cells transfected with the indicated siRNAs were subjected to an in vitro elongase assay as in A using the indicated acyl-CoA (50 μM) and 0.04 μCi [14C]malonyl-CoA. Values represent the mean ± SD from three independent experiments. Statistically significant differences are indicated (*p < 0.05; t test). (D) HEK 293T cells were transfected with a pcDNA3 HA-1 control vector, or a pcDNA3 HA-CERS2 or pcDNA3 HA-CERS4 plasmid, and with a pCE-puro 3xFLAG-ELOVL1 plasmid or pCE-puro 3xFLAG-1 control vector. Total cell lysates were prepared from the transfected cells and solubilized with 1% Triton X-100. Total lysates (Left) or proteins immunoprecipitated with anti-FLAG M2 agarose (Right) were subjected to immunoblotting with anti-FLAG or anti-HA antibodies. IP, immunoprecipitation; IB, immunoblotting; Total, total cell lysates.