Figure 2.

LCFA uptake assays. (A) COS cells were cotransfected using the DEAE-dextran method with the mammalian expression vectors pCDNA-CD2 either alone (Control) or in combination with one of the FATP-containing expression vectors (pCDNA-mmFATP1, pCDNA-mmFATP2, or pCMV-SPORT2-mmFATP5) as described in Materials and Methods. COS cells were gated on FSC and side scatter, and the results shown represent >10,000 cells. Cells exhibiting >300 CD2 fluorescence units (vertical line) representing ≈15% of all cells were deemed CD2 positive. (B) As in A, COS cells were cotransfected with pCDNA-CD2 either alone (Control) or in combination with one of the FATP-containing expression vectors (pCDNA-mmFATP1, pCDNA-mmFATP2, pCMV-SPORT2-mmFATP5, or pCDNA-ceFATPb). The mean BODIPY-FA fluorescence of the CD2-positive cells is plotted; results shown represent the average of three experiments, each consisting of >50,000 COS cells. Note that a logarithmic scale is used on the ordinate. (C) The full-length coding region of mtFATP (squares) and a control protein (TFE3; circles) were subcloned into the inducible, prokaryotic expression vector pET (Novagen). Expression was induced (solid symbols) with 1 mM isopropyl β-d-thiogalactoside for 1 hr or cells were left uninduced (open symbols). Data points were done in triplicate and counts were normalized to the number of bacteria as determined by OD₆₀₀.