[Construction of eukaryotic plasmid expression Hsf4b and phosphorylation of Hsf4b by MAP kinase P38].

AIM

Ton construct eukaryotic plasmid expressing Hsf4B and to investigate Hsf4b is phosphorylated by MAP kinase P38:

METHODS

The total RNA of human heart tissues were prepared. Hsf4b cDNA were then synthesized with RT-PCR. The PCR products were digested with Kpn I and EcoR I and subcloned into pcDNA3.0, pcDNA-Flag-Hsf4b was transfected into HEK293T cells. The expression of Hsf4b was testified with Western blotting. The interaction between Hsf4b and P38 was assayed by immunoprecipitation. In vivo pull down GST demonstrated that Hsf4B (196-493) could interact with P38, P38 phosphorylation of Hsf4b were testified with Kinase assay.

RESULTS

We subcloned the human cDNA of Hsf4b into eukaryotic expression vectors pcDNA3 and PEBG, and Hsf4b was overexpressed in HEK293T cells. Further studies demonstrated that Hsf4b could interact with and phosphorylated by MAP kinase P38.

CONCLUSION

Hsf4b could interact with and phosphorylated by MAP kinase P38. Our results will provide more evidence for understanding the signal regulation of Hsf4b transcription activity during lens development.