Skip to main content
. Author manuscript; available in PMC: 2018 Aug 8.
Published in final edited form as: Dev Growth Differ. 2009 May;51(4):403–410. doi: 10.1111/j.1440-169X.2009.01097.x

Fig. 5.

Fig. 5.

Purified Nkx2–5 protein binds to the Nkx2–5 ci-elements within the Tolloid-like 1 (Tll-1) promoter. (A) Electrophoretic mobility shift assay of the proximal Tll-1 Nkx2–5 site, the Mut site or a nonsense site with protein extracts containing Nkx2–5 grown in COS-1 cells. The sequences of the oligonucleotides are shown at the bottom of the figure. Changes in sequence from the mouse wild-type are indicated in red. Lane 1, Tll-1 proximal Nkx2–5 free probe. Lane 2, Tll-1 proximal Nkx2–5 site incubated with nuclear extract purified from COS-1 cells transfected with pc DNA 3.1 control plasmid. Lane 3, Tll-1 proximal Nkx2–5 site incubated with Nkx2–5 protein. Lane 4, Tll-1 proximal Nkx2–5 site incubated with Nkx2–5 protein and 1 × cold Tll-1 proximal Nkx2–5 site competitor. Lane 5, same as Lane 4 except with 100× cold competitor. Lane 6, Mut sequence incubated with Nkx2–5 protein. Lane 7, Nonsense sequence incubated with Nkx2–5 protein. Lane 8, Same as Lane 3 control antibody. Lane 8 Same as Lane 3 incubated with anti-Nkx- 2–5 antibody showing supershift. (B) Electrophoretic mobility shift assay of the medial Tll-1 Nkx2–5 site, the Mut site or a nonsense site with protein extracts containing Nkx2–5 grown in COS-1 cells. The sequences of the oligonucleotides are shown at the bottom of the figure. Changes in sequence from the mouse wild-type are indicated in red. Lane 1, Tll-1 medial Nkx2–5 free probe. Lane 2, Tll-1 medial Nkx2–5 site incubated with nuclear extract purified from COS-1 cells transfected with pc DNA 3.1 control plasmid. Lane 3, Tll-1 medial Nkx2–5 site incubated with Nkx2–5 protein. Lane 4, Tll-1 medial Nkx2–5 site incubated with Nkx2–5 protein and 1 × cold Tll-1 medial Nkx2–5 site competitor. Lane 5, same as Lane 4 except with 100 × cold competitor. Lane 6, Mut sequence incubated with Nkx2–5 protein. Lane 7, Nonsense sequence incubated with Nkx2–5 protein. (C) Electrophoretic mobility shift assay of the distal Tll-1 Nkx2–5 site, the Mut site or a nonsense site with protein extracts containing Nkx2–5 grown in COS-1 cells. The sequences of the oligonucleotides are shown at the bottom of the figure. Changes in sequence from the mouse wild-type are indicated in red. Lane 1, Tll-1 distal Nkx2–5 free probe. Lane 2, Tll-1 distal Nkx2–5 site incubated with nuclear extract purified from COS-1 cells transfected with pc DNA 3.1 control plasmid. Lane 3, Tll-1 distal Nkx2–5 site incubated with Nkx2–5 protein. Lane 4, Tll- 1 distal Nkx2–5 site incubated with Nkx2–5 protein and 1 × cold Tll-1 distal Nkx2–5 site competitor. Lane 5, same as Lane 4 except with 100 × cold competitor. Lane 6, Mut sequence incubated with Nkx2–5 protein. Lane 7, Nonsense sequence incubated with Nkx2–5 protein.