Figure 9

Two-hybrid interaction analysis of oligomer formation. Isoform 1, 2 and 3 and ΔN-isoform 2 were cloned in the Bait vector (see Methods). Isoform 3 was cloned in the Target vector. Results obtained with the combination of all isoforms cloned into both vectors as well as the complete set of controls cells and conditions are presented in Additional file 2. Growth tests were performed on solid media in absence (non-selective) or presence (selective) of the histidine biosynthesis inhibitor 3'-AT at a concentration of 5 mM. Expression of the cloned ACSL6 proteins was placed under the control of an IPTG-inducible promotor and tests were performed without (not induced) or with (induced) 30 μM IPTG (see methods). As seen on the control plates and as expected, the E. coli cells transformed with empty Bait and Target vectors (-/-) grew in absence of the drug but failed to grow in its presence and, cells transformed with the Bait and Target positive controls (+/+; dimerization domain of Gal4 with interacting domain of Gal11) grew in presence of the drug only when IPTG (induced condition) was present in the medium. Plates on the left side of the figure represent the experimental plates with cells transformed with isoform 3 Target construct and with one of the 4 different Bait constructs, as indicated. Note that, as expected, all cells transformed with the 4 different Bait constructs and with the empty Target vector grew in absence of the drug but not in its presence.