Abstract
MAGE-G1 is a protein plays role in the early process of neurogenesis. However, the fundamental roles MAGE-G1 played in neurogenesis have not yet been completely understood. Finding the partners MAGE-G1 interacting with will surely contribute to the function study of MAGE-G1. In this study, using Stable Isotope Labeling by Amino acids in Cell culture-immunoprecipitation quantitative proteomics, we screened the interacting proteins of MAGE-G1 during retinoic acid -induced neuronal differentiation of P19 cells and firstly found that FSCN1 and VIME were potential novel MAGE-G1-interacting proteins. Then, the interaction between overexpressed MAGE-G1 and FSCN1 or VIME was validated by GST-pull down assay in bacteria and by co-immunoprecipitation assay in COS7 cells. Endogenous co-immunoprecipitation assay further confirmed that MAGE-G1 interacted with FSCN1 or VIME in P19 cells after a 6-day retinoic acid-induced neuronal differentiation. Those results provide a functional linkage between MAGE-G1 and FSCN1 or VIME and may facilitate a better understanding of the fundamental aspects of MAGE-G1 during neurogenesis.
Highlights
As one of Necdin homologous genes, Mage-g1 gene has been mapped to proximal chromosome 15q4
A P19 cell line which stably expressed the Flag-tagged mouse MAGE-G1 was generated, and the specific MAGE-G1 interactome formed in P19 cells during retinoic acid (RA)-induced neuronal differentiation was screened using the SILAC-based quantitative proteomic approach
P19 cells were treated with RA for 6 days to induce neuronal differentiation, and the proteins were extracted from each group and mixed in a ratio of 1:1 based on the total protein mass
Summary
As one of Necdin homologous genes, Mage-g1 gene ( designated Necdin-like 2) has been mapped to proximal chromosome 15q4. A number of techniques are used to screen unknown interacting proteins, which include the yeast two-hybrid system, pull-down assays, as well as tandem affinity purification (TAP)[9,10]. Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-immunoprecipitation quantitative proteomics provides a useful tool to overcome the disadvantages mentioned above[11]. Using this method to screen interacting proteins, specific partners appear as isotopically heavy, while non-specific interaction partners appear as a mixture of isotopically light and heavy at a 1:1 ratio. The interactions were further validated by both exogenous and endogenous co-immunoprecipitation assay
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