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. 2010 Jul 27;1(4):1–10. doi: 10.1038/ncomms1041

Figure 2. Hand1 binds to a region proximal to the Tβ4 promoter in vivo.

Figure 2

Regions of the Tβ4 sequence containing either the E-box (F1–R1; forward (F) and reverse (R) primer sequences are underlined with arrowhead for orientation) or the Thing1 box (F2–R2 primer sequences as indicated) were PCR-amplified using indicated primers (a). Chromatin containing the E-box and the Thing1 box was individually immunoprecipitated from wild-type (WT) embryoid bodies but not from Hand1-null embryoid bodies as confirmation of antibody specificity (b). Hand1 binds to the Tβ4 promoter in the E8.5 embryo but only weakly at the E-box of the promoter at E11.5. A 469-bp product containing both binding sites (F1–F3 primer sequences as indicated) could be amplified from both EBs and E8.5 embryos under PCR conditions of reduced stringency. Lanes 1, 2 and 3 refer to input, no antibody control and Hand1 antibody, respectively. Specificity of the antibody used for ChIP was confirmed by western blot analysis of E8.5 WT (+/+) and Hand1-null (−/−) embryos (c); black arrowhead highlights specific Hand1 band; ns, nonspecific.