Supplementary Figure 2: The PAX3-MAML3 fusion protein localizes to the nucleus and binds DNA.

(a) Protein blots were performed with antibody to PAX3 or tubulin on lysates of HEK293T cells expressing pcDNA3 (Co), Flag-PAX3-MAML3 (P-M) or HA-PAX3. (b) Immunofluorescence images showing nuclear expression of Flag-PAX3-MAML3 in C2C12 cells. Cells were counterstained with phalloidin to detect cytoplasmic F-actin and with DAPI to detect DNA. (c) Electrophoretic mobility shift assay (EMSA) of Flag-PAX3-MAML3 binding to double-stranded DNA containing a PAX3-binding element. Lysates from COS-7 cells expressing Flag-PAX3-MAML3 or HA-PAX3 were incubated with a ³²P-labeled PAX3 DNA probe in the presence or absence of 100-fold excess of the wild-type (WT) probe, a mutated (Mut) probe, control IgG, antibody to HA or antibody to Flag. The arrow points to PAX3-MAML3–DNA complexes. The arrowhead indicates PAX3-DNA complexes. The asterisk indicates free probe. (d) Transient transcription assays demonstrating the effect of PAX3-MAML3 on a Notch reporter, Hes5p-Luc, in C2C12 cells in the presence or absence of the Notch1 intracellular domain 1 (NICD1). *P < 0.01, **P < 0.0001 relative to control in unpaired t tests.