Genome-wide maps of gV sPLA2 binding sites in Raw 264.7 cells

We have searched genome-wide binding sites of mouse group V secretory phospholipase A2 (gV sPLA2) in Raw 264.7 cells. Since we previously had technical problems in immunoblotting analysis and immunoprecipitation of the gV sPLA2 protein due in part to unavailability of a specific antibody against mouse gV sPLA2 and other issues, we used a monoclonal antibody against the Myc-tag to analyze gV sPLA2 knockdown (KD) RAW 264.7 cells with re-constitutive expression of Myc-tagged gV sPLA2 or Myc-tagged gV sPLA2-H48Q, lacking catalytic activity, as well as gV sPLA2 KD RAW 264.7 cells transfected with empty vector. Among the significant peaks, the region corresponding to -375 ~ +6 from the transcriptional start site of the Pgk1 (phosphoglycerate kinase 1) gene was the strongest binding peak in the nucleus of both gV sPLA2 KD cells expressing Myc-tagged gV sPLA2 and gV sPLA2 KD cells expressing Myc-tagged gV sPLA2-H48Q. Meanwhile, the Pgk1 gene was not identified as a significant peak in the nucleus of gV sPLA2 KD cells transfected with empty vector. Examination of binding sites of gV sPLA2 protein in Raw 264.7 cell line.

本研究在Raw 264.7细胞中检测了小鼠V型分泌型磷脂酶A2（group V secretory phospholipase A2, gV sPLA2）的全基因组结合位点。此前由于缺乏针对小鼠gV sPLA2的特异性抗体等技术问题，我们无法顺利开展gV sPLA2蛋白的免疫印迹分析与免疫沉淀实验；因此我们使用针对Myc标签（Myc-tag）的单克隆抗体，对三类细胞进行检测：分别为重组表达带有Myc标签的gV sPLA2的gV sPLA2基因敲低（knockdown, KD）Raw 264.7细胞、重组表达带有Myc标签且催化活性缺失的gV sPLA2-H48Q的gV sPLA2 KD Raw 264.7细胞，以及转染空载体的gV sPLA2 KD Raw 264.7细胞。在检测得到的显著结合峰中，对应Pgk1（磷酸甘油酸激酶1，phosphoglycerate kinase 1）基因转录起始位点上游-375至下游+6的区域，在表达Myc标记的gV sPLA2的gV sPLA2 KD细胞与表达Myc标记的gV sPLA2-H48Q的gV sPLA2 KD细胞的细胞核中均为最强结合峰。与此同时，在转染空载体的gV sPLA2 KD细胞的细胞核中，并未检测到Pgk1基因区域存在显著结合峰。本研究针对Raw 264.7细胞系中gV sPLA2蛋白的结合位点开展了相关检测。