Figure 1. ELOVL4 is not involved in the elongation of eicosapentaenoic acid (EPA) or docosapentaenoic acid (DPA). HEK 293T cells were transfected with a vector (pCE-puro HA-1) or a plasmid encoding the indicated HA-tagged human ELOVL protein. A: Total membrane proteins (3 μg protein) prepared from the transfected cells were separated by sodium dodecyl sulfate PAGE (SDS–PAGE), and detected by immunoblotting with anti-HA antibodies. Uniform protein loading was demonstrated by immunoblotting with anti-calnexin antibodies. B, C: Total membrane proteins (20 μg of protein) were incubated for 30 min at 37 °C with 50 μM acyl-CoA(C24:0-CoA or C26:0-CoA; B) or with 50 μM FAs (EPA or DPA), 10 mM ATP, and 200 μM CoA (C) in the presence of 1 mM NADPH and 0.075 μCi [¹⁴C]malonyl-CoA. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal phase thin layer chromatography (TLC), followed by detection and quantification by a bioimaging analyzer BAS-2500. Values presented represent the mean±standard deviation (SD) from three independent experiments. Statistically significant differences compared to vector-transfected cells are indicated (**p<0.01; t-test). Abbreviations: IB represents immunoblotting, 4 represents ELOVL4; 4Δ represents ELOVL4ΔC; 2 represents ELOVL2; 5 represents ELOVL5.