Fig. 3. Embryonic gel shift analysis of BMPRE. (A,B) Dissected regions of HH stage 6-8 and 24 (day 3) chick embryos are depicted as boxed regions of whole mount chick embryos stained by in situ hybridization for Nkx2.5 mRNA. Whole-cell extracts were made from either anterior lateral plate (ALP) or posterior primitive streaks (PPS) dissected from stage 6-8 chick embryos (A), or from hearts (Hrt) from stage 24 (day 3) chick embryos (B). (C) EMSA oligomers derived from 200 bp BMPRE enhancer sequence (see Fig. 2). Numbered and colored horizontal lines show extent of 30 or 40 bp double stranded oligomers used for gel shift assays. (D) Gel shifts obtained with anterior lateral plate (A), posterior primitive streak (P) or 3 day heart whole cell extracts. Discrete shifts were found for eight sites labeled A-E. Sites A1-A3 (nucleotides 1-30), lanes 1, 2, 21; site B (nucleotides 30-60 and 45-75), lanes 5-8, 23, 24; site C (nucleotides 60-100), lanes 9, 10, 25; site C' (nucleotides 100-140), lanes 13, 14, 27; site D (nucleotides 120-160), lanes 15, 16, 28; site E (nucleotides 160-200), lanes 19, 20, 30. Multiple shifts seen with Site A oligo in different extracts are labeled separately A1-3 to the left of the autoradiogram, and are as discussed in the text.