Identification of immunoglobulin switch (S) region binding proteins by insertional ChIP (iChIP)

We used insertional ChIP (iChIP) based approach to identify proteins that bind to mouse immunoglobulin switch (S) region. To perform iChIP, we inserted an 8X-repeat of the LexA-binding element (LexA-BE) downstream of the Sα region in CH12F3-2A cells, a mouse B-cell line. The DNA-binding domain and dimerization domain of the LexA protein fused with a FLAG tag and a nuclear localization signal (NLS) was expressed in WT (FNLDD-alone) or Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The resultant cells were subjected to stable isotope labeling with amino acids in cell culture (SILAC), stimulated with CIT (CD40L, IL4, and TGFβ), immunoprecipitated with an anti-FLAG antibody, and subjected to LC-MS/MS analysis. The identities of the deposited data are as follows: F: WT (FNLDD-alone); D: Sα-engineered (FNLDD-Sα-LexA).