Figure 3. DUX4c protein expression in myoblasts.

(A) Transcription/translation in vitro in a rabbit reticulocyte lysate in the presence T7 RNA polymerase and [³⁵S]-cysteine with genomic fragments encoding DUX4 (lane 1) or DUX4c (lane 2) cloned in pCIneo. 52 kDa-DUX4 (white arrow) and 47 kDa-DUX4c (black arrow) are detected by autoradiography after 10% PAGE-SDS. (B–C) 30 µg total proteins extracted from primary myoblast were analysed by 4–12% PAGE-SDS and Western blot with the indicated primary antibodies, appropriate secondary antibodies coupled to HRP and the ECL kit. α-Tubulin was the loading control. (B) Competition: a 5-fold excess of DUX4c antigenic peptide was pre-incubated (+) or not (−) with the serum raised against DUX4c or cadherin as indicated. (C) Extracts were prepared either from proliferating myoblasts or 2 (d2) or 6 (d6) days after induction of differentiation. (D) DUX4c (red) was detected by immunofluorescence in nuclei of myoblasts and myotubes 2 (d2) and 6 (d6) days after inducing differentiation. a' and b' correspond respectively to two enlarged nuclei from d2 and d6 (white boxes in a and b). The labeling is weakened after competition with the immunogenic peptide (c). Troponin T (green) is a myotube differentiation marker. Myoblasts not fused to myotubes express DUX4c (red nuclei) but not troponin T. Bar corresponds to 20 µm. (E) 30 µg protein extracted from primary myotubes were analyzed by Western blot as in (C). (F) Densitometric scanning of the film shown in (E): DUX4c expression levels were normalized to α-Tubulin (relative absorbance units). C: control, F: FSHD, D: DMD.