Fig. 9. Tbx20 cooperatively activates transcription. (A) Activation of various reporter constructs by Tbx20, Tbx5 or Tbx1 expression constructs co-transfected into 10T1/2 cells. Reporter constructs used are Nkx2-5-luciferaseFL (Nkx-up), Pitx2-luciferase (Pitx2), Fgf10-luciferase (Fgf10), Myl7-luciferase (b-MHC), Gja5-luciferase (Cx40) and Nppa-luciferase (ANF). (B) Activation of the Mef2c-lacZ #3 reporter by Isl1, Gata4, Nkx2-5 or Foxh1 expression constructs, with (+, ++) or without (–) a Tbx20 expression construct. Red bars indicate 250 ng of the indicated expression construct, blue bars indicate 500 ng of the expression construct. +, 250 ng Tbx20 expression construct; ++, 500 ng Tbx20 expression construct. (C) Alignment of mouse (mm) and human (hs) sequence of part of the Nkx2-5 enhancer that comprises the Isl1-binding site (Isl1, red) and the Gata-binding site (Gata, blue). (D-H) Transgenic mouse embryos (E9.75) carrying the Nkx2-5-lacZ transgene (Nkx2-5 wt, D,F), or the Nkx2-5-lacZmutIsl1 transgene (Nkx2-5 Isl1 mut, E,G) shown as whole-mounts (D,E) or sections of the whole-mount stained embryos (F,G). Arrowhead shows expression in pharynx. For Nkx2-5 wild type, nine out of 12 Southern blot-positive embryos expressed lacZ strongly in the pattern shown; for Nkx2-5 Isl1 mut, 11 out of 14 Southern blot-positive embryos expressed lacZ weakly in the pattern shown, three were completely negative. (H) Activation of the Nkx2-5-luciferase FL reporter by Tbx20, Gata4, Isl1 or activated Alk3 (aAlk3) expression constructs, co-transfected singly or in combination in 10T1/2 cells. (I) Co-immunoprecipitation shows interactions between Isl1 and Myc-Tbx20 (m-T20), but not FLAG-Gata4 (F-G4), myc-Tbx1 (m-T1) or FLAG-Tbx5 (F-T5). Bottom two panels show 10% input.