Figure 6.

Brn1/2 regulate pro-neural genes Ngn2 and Tbr2 in vitro. (A and B) The design of Ngn2 and Tbr2 reporters is similar to those described previously (Shimojo et al. 2008; Ochiai et al. 2009). (C″ and G) N2a cells at 24-h post-transfection and serum starvation labeled for Tbr2 (C″ and D), Ngn2 (E″ and G), or immunoblotted (H) for Tbr2. Mock transfected cells do not express either Tbr2 (C″ and C) or Ngn2 (E″ and E) endogenously. Sporadic Brn2-transfected cells (but not all) express detectable levels of Tbr2 (D″ and D), and sporadic Pax6-transfected cells (but not all) express detectable levels of Ngn2 (F″ and F); Brn2 does not stimulate endogenous Ngn2 expression under these conditions (G″ and G). Immunoblot analysis for Tbr2 demonstrates similar findings (H). (I and J) Ngn2 and Tbr2 reporter activity showns strong activation by Brn1/2 alone and in combination with known upstream proneural regulators. All luciferase values were normalized to the transfection condition generating the maximal activity.