Figure 2.

Expression of SDS-resistant, potential multimers upon transfection of DUX4 construct plasmids. (A) SDS-PAGE and immunoblotting were used to analyze proteins expressed in HeLa cells at 24 h after transfection with the indicated DUX4 pCS2(+)-V5 plasmids. On the upper blot, proteins were detected with a mAb that reacted with the V5 epitope on the C-terminus of each construct. In this figure and all following figures, each band marked with a single asterisk is the size predicted for a monomeric protein expressed from the corresponding cDNA construct, whereas bands marked with double and triple asterisks are the sizes expected for potential dimers and trimers, respectively. The potential dimer band was most noticeable for DUX4-S (lane 2). Numbers to left of blot indicate molecular mass in kDa. (B) Densitometry of immunoblots was used to determine the percentage of protein expressed from each construct that was the size of a potential dimer. Linear curve-fitting was used to derive the solid line (y = 73.6-0.053x, R = 0.90). The percentage of potential dimers decreased as the cDNA insert became longer. (C) In a separate experiment, HEK293 cells were transfected with the DUX4-FL plasmid for 48 h and the resulting proteins were detected by immunoblotting either with mAb E55, which is specific to an epitope in the C-terminal portion of DUX4-FL²², or with anti-V5 as indicated. Two major bands reacted with both antibodies, one the size of the expected monomer (single asterisk) and a second the size of a potential dimer (double asterisk). Also seen were potential higher order multimers, as well as intermediate and smaller bands that could be proteolytic products. This result shows that the SDS-resistant, potential multimers included the E55 (endogenous DUX4-FL) and V5 (tag) epitopes. MW = markers of protein molecular mass shown in kDa.