Fig. 8. Analysis of fatty acid uptake. (A) FACS of COS cells cotransfected with the red fluorescent tdimer protein. This enables the correlation of expression with the amount of the fluorescent fatty acid analog B₁₂-FA taken up. Both FATP4 (r=0.93) and ACSL1 (r=0.71) enhance fatty acid uptake depending on their relative level of expression. The S247A mutant FATP4 protein (serine 247 critical for AMP binding changed to alanine) is not significantly different from control cells expressing only tdimer. (B) Acyl-CoA synthetase activity (in pmol oleoyl-CoA/minute/µg protein) determined from the lysates of COS cells. Controls for endogenous ACS activity are transfected with empty plasmid (pcDNA3) or the S247A-FATP4 mutant. The ACS activities should be considered qualitative rather than absolute because ACSL1 and FATP4 showed inverse susceptibilities towards the detergent used for solubilization (see Materials and Methods). (C) Oleate uptake of COS cells (pmol oleate/µg protein after 5 minutes). (D) Lipid analysis after oleate uptake. After 5 minutes, 87-92% of oleate is already metabolized into phospholipids and neutral lipids. The level of the remaining free oleate is only 5-8%.