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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Dev Biol. 2013 Sep 10;383(1):10.1016/j.ydbio.2013.09.006. doi: 10.1016/j.ydbio.2013.09.006

Fig. 2. Sequences in the Myf5 ES enhancer and promoter are required for Pax3 synergy with Gli2 and Zic1.

Fig. 2

Fig. 2

Fig. 2

Fig. 2

A. Labeled probe containing a consensus Pax3 binding site from the −58/−56 kb distal Myf5 enhancer (11) was mixed with nuclear extracts from 10T1/2 cells overexpressing Pax3-HA, and analyzed via gel-shift assays. Antibodies (αHA, lane 2) or competitor oligos (Pax3 consensus motif, lane 3; wt HD motif in Myf5 promoter, lane 4, and mt HD motif in Myf5 promoter, lane 5) are indicated. The complex containing Pax3-HA bound to the probe (supershifted with αHA in lane 2) is labeled. Arrowhead indicates free probe. B. A luciferase reporter plasmid containing the Myf5 ES enhancer and promoter with a mutation in the promoter HD motif (HD-mt) was transiently transfected into 3T3 cells with or without expression plasmids for Zic1, Gli2, or Pax3. C. A luciferase reporter plasmid containing 8 consensus Gli motifs upstream of the thymidine kinase (TK) promoter was transiently transfected into 3T3 cells with or without expression plasmids for Zic1, Gli2, or Pax3. D. A luciferase reporter plasmid containing the Myf5 ES enhancer and promoter with a mutation in the ES enhancer Gli motif (Gli-mt) was transiently transfected into 3T3 cells with or without expression plasmids for Zic1, Gli2, or Pax3. For B–D, cells were harvested and assayed, and data was analyzed as in Fig. 1. Asterisk in D indicates synergy between Zic1 and Pax3 (p < 0.05, one-tailed, one-step t-test comparing activity of factors in combination to the sum of individual factors).