Identification of interaction partners of PRMT6

To identify transcription factors of PRMT6 which could recruit PRMT6 to target genes, we used affinity purification of avi-tagged PRMT6 in combination with stable isotope labeling of amino acids in cell culture (SILAC) based mass spectrometry. For this, K562 cells were transduced with a lentiviral co expression vector for the BirA-ligase and avi-PRMT6 with a 21 amino acid taq, which is biotinylated in the cells. Cells expressing the BirA-ligase only served as a control. Avi-PRMT6 cells were grown in heavy SILAC medium and control cells in light SILAC medium for seven passages. Nuclear extracts were prepared from 1x108 cells and subjected to avi-PRMT6 affinity purification using magnetic streptavidin beads. Subsequently, the proteins were eluted from the beads. The eluates from the avi-PRMT6 and the control were mixed in a one to one ratio. Subsequently, we performed quantitative mass spectrometry (MS)-based analysis of the PRMT6 interactome.

为鉴定能够将PRMT6招募至靶基因的转录因子，我们将avi标签标记的PRMT6亲和纯化技术，与基于细胞培养氨基酸稳定同位素标记（Stable Isotope Labeling of Amino acids in Cell culture, SILAC）的质谱技术相结合开展实验。
为此，我们使用携带BirA连接酶与带有21个氨基酸标签的avi-PRMT6的慢病毒共表达载体转导K562细胞，该标签可在细胞内被生物素化。仅表达BirA连接酶的细胞作为对照。
将表达avi-PRMT6的细胞置于重链SILAC培养基中培养，对照细胞置于轻链SILAC培养基中连续传代7次。
从1×10^8个细胞中制备核提取物，使用链霉亲和素磁珠进行avi-PRMT6亲和纯化。随后将结合在磁珠上的蛋白洗脱下来。
将avi-PRMT6实验组与对照组的洗脱液按1:1比例混合。
此后，我们对PRMT6互作组开展基于定量质谱（Mass Spectrometry, MS）的分析。