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


CONCLUSIONS
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.
Ma
Zheng-yi
ZY
Key laboratory of Cell and Molecular Immunology, Medical college of Henan University, Kaifeng, China.
Zhang
Jun
J
Li
Xue-li
XL
Xi
Zi-ming
ZM
Hu
Yan-zhong
YZ
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D000076249:Heat Shock Transcription Factors; D006801:Humans; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C102948:HSF4 protein, human; D000076249:Heat Shock Transcription Factors; D014157:Transcription Factors; D048051:p38 Mitogen-Activated Protein Kinases