Mtss1抗体制备及小脑中不同亚型磷酸化水平的检测

Antibody Preparation and Tyrosine Phosphorylation Level of Mtss1 in the Different Isoforms of Rat Cerebellum

肿瘤转移抑制蛋白(Metastasis suppressor1,Mtss1),又名肿瘤转移消失蛋白(Missing in metastasis,MIM)在小脑神经元发育中受到调控,依次表达两种亚型:包含Src磷酸化位点的广泛表达亚型;含Src磷酸化位点片段经RNA剪接去除的神经元特异亚型.为检测这两种Mtss1亚型的酪氨酸磷酸化水平是否因Src磷酸化位点的去除存在明显区别,制备了灵敏度较高并可特异性沉淀外源和内源表达的Mtss1的兔多克隆抗体,对发育时期与成年大鼠小脑内源Mtss1酪氨酸磷酸化水平进行检测,发现成年后的Mtss1与出生后发育时期的Mtss1均发生明显的酪氨酸磷酸化,表明剪接去除包含有Src磷酸化位点中的神经元特异亚型中,还有其他的酪氨酸残基被磷酸化,提示其他酪氨酸磷酸化激酶信号通路对Mtss1神经元亚型的调控作用.

Metastasis suppressor 1 （Mtss1）, namely missing in metastasis （MIM）, has been shown to be developmentally regulated in the cerebellar development. Two isoforms have been presented in the different developmental stages of cerebellum： one isoform prevalent in the developmental stage is ubiquitous expressed type, containing important Src phosphorylation sites; another isoform expressed only in the adult cerebellum, so-called neural isoform, have no Src phosphorylation site due to alternative splicing. To investigate whether there was obvious difference of the tyrosine phosphorylation level between these two isoforms, rabbit polyclonal to Mtssl which could be applied to immunoprecipitaion were prepared and then the cerebellar extracted from postnatal developmental and adult rat were used for immunoprecipitation and consequently Western blot by phosphorylation antibody. As the result, obvious tyrosine phosphorylation of Mtssl was detected from both stages. It suggested the presence of phoshphorylated tyrosine residues other than Src phosphorylation sites and neural isoform of Mtssl might be regulated by multiple tyrosine phosphorylation kinase involved signaling pathways.