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Fig. 3. The CREB protein is involved in the expression of Nkx2.5 downstream of p38 MAPK. (A) Stage 16 embryos were immunostained using an antibody to phospho-CREB (S133) (left) or analyzed by in situ hybridization with a probe to Nkx2.5 (right). (B) CREB AMO was injected to embryos, and gene expression was assessed in three cardiac explant assays; DMZ explants (left), AC explants from Alk4-expressing embryos (middle) and VMZ explants from LiCl-treated embryos (right). At stage 15 or stage 23, total RNA was isolated and RT-PCR analysis was performed. (C) Transcripts encoding VP16-CREB were injected to one of two blastomeres (right panel) or to two of two blastomeres (left panel). The expression of Nkx2.5 was detected by in situ hybridization (right panel) or by RT-PCR analysis on VMZ explants (n = 18) (left panel). (D) Different combinations of transcripts encoding activated MKK6 and CREB AMO were injected to embryos and VMZ explants were isolated and grown to stage 14 (left panel). Different combinations of transcripts encoding VP16-CREB and p38α AMO were injected to embryos and DMZ explants were isolated and grown to stage 14 (right panel). Total RNA was isolated and RT-PCR analysis was performed. |
