Fig 6. p65 Arg¹⁷⁴ methylation is present on the CXCL11 promoter following costimulation.

(A) EC were depleted of endogenous p65 and reconstituted with wild type or Arg¹⁷⁴Lys mutant p65. Cells were stimulated with TNF plus IFN-γ for 30 minutes and lysed in RIPA buffer. Wild type and mutant p65 was immunoprecipitated using the FLAG epitope or IgG. Immunoprecipitates were immunoblotted for p65 and for the SDMA modification. (B-C) Cells were reconstituted with N-FLAG wild type, Arg³⁰Lys, Arg³⁵Lys, and Arg¹⁷⁴Lys p65 constructs, and simultaneously exposed to TNF and IFN-γ. ChIP was performed using anti-FLAG antibody. Sequential ChIP (Re-ChIP) was carried out with addition of normal rabbit serum or anti-SYM10 antibody. Following crosslink reversal, DNA was purified and promoter fragment content was quantitated using qRT-PCR relative to input.