Figure 8

Detection of isoforms in the membrane of E. coli. ACSL6 forms were expressed in E. coli. Membrane and soluble fractions were obtained as described in the methods section. Proteins were separated on denaturing SDS-PAGE 7.5% gel and stained with coomassie-blue. The molecular weight standard is shown on the right. Position of the monomer and dimer species is indicated with an arrow. A. Proteins present in the membrane (lane 1 to 5) and in the soluble (lane 6 to 9) fractions of E. coli carrying the vector and the different isoforms, as indicated, are shown. B. Protein present in membrane fractions of E. coli carrying the vector, isoform 1 (Y-Gate), ΔN-(Y-Gate), isoform 2 (F-Gate), isoform 3 (no-Gate), ΔN-(F-Gate), ΔGate and, ΔN-ΔGate are shown. C. Histogram of the ratios of the intensity value of the slow to the fast migrating bands of isoform 1 and isoform 2 and of their respective N-terminus truncated version (ΔN-Y-Gate and ΔN-F-Gate) is shown. Density of bands with molecular mass of ≈75 kDa (monomer) and ≈140 kDa (dimer) detected on lane 2, 3, 4 and 6 of the gel shown on panel B were quantified using QuantityOne program (Bio-Rad). Intensity values obtained in lane 1 (E. coli proteins only) at the same position on the gel, taking into account the slight difference of migration of the different bands, were subtracted to the intensity value of each of the bands of each of the isoforms and constructs.