Figure 4. ELOVL4 mutant oligomerizes with wild-type ELOVL4

Whole cell lysates prepared from cells transfected with EGFP- or V5-tagged ELOVL4 constructs were first subjected to CN-PAGE in the first dimension to separate the multiprotein complexes in accordance with their molecular mass and state of aggregation. Following this first dimension separation, each sample lane (0.5 cm in width) of the first dimension native gel was cut out and equilibrated with Laemmli Buffer (62.5 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 2% (w/v) DTT, 5% (v/v) glycerol) for 30 min at ambient room temperature. This SDS treatment is to break apart aggregates into single polypeptides that should migrate in the second dimension gel according to the molecular mass of the polypeptide. The gel strip was placed horizontally (at right angles to the first dimension) at the usual position for the stacking gel in the second dimension 10% SDS-polyacrylamide gel and overlaid with 0.5% agarose in Laemmli buffer. After solidification of the agarose, the second dimension electrophoresis was conducted, and immunoblot analyses were performed with GFP or V5 epitope specific antibodies according to standard protocols. A: GFP-vector. B: GFP-Ewt. C: GFP-Emut. D: GFP-Ewt and GFP-Emut. E: GFP-Ewt and V5-E mut. F: V5-E wt and GFP-Emut. G: V5-E wt. H: V5-E wt and GFP-Emut.