Fig. 4.

CITED2 is essential for post-translational modifications of NCL. a HEK293T cells were transfected with pcDNA or CITED2 and treated with Leptomycin B (200 nM), and the cell lysates were fractionated to cytosolic and nuclear components. The cell fractions were immunoblotted with the indicated antibodies. b HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2 or si-CITED2. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with the indicated antibodies. c HEK293T cells were transfected with Myc-PRMT5 and/or si-PRMT5. Cell lysates were immunoprecipitated with anti-dimethyl arginine antibody and precipitated NCL was immunoblotted. d HEK293T cells were transfected with HA-p300. Cell lysates were immunoprecipitated with anti-acetyl lysine antibody and precipitated NCL was immunoblotted. e, f HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2 or si-CITED2. Cell lysates were immunoprecipitated with anti-dimethyl arginine or anti-acetyl lysine antibody and precipitated NCL was immunoblotted. g, h HEK293T cells were cotransfected with the indicated plasmids and siRNAs. Cell lysates were immunoprecipitated with anti-dimethyl arginine or anti-acetyl lysine antibody and precipitated NCL was immunoblotted. i HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2, and the cell lysates were fractionated to cytosolic and nuclear components. The cell fractions were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. j The ERG–CITED2–PRMT5/p300–NCL pathway in prostate cancer. ERG is upregulated due to the TMPRSS2–ERG gene fusion and transactivates the CITED2 gene in prostate cancer cells. Overexpressed CITED2 induces the methylation and acetylation of NCL in the nucleus by recruiting PRMT5 and p300, then modified NCL is translocated to the cytoplasm. All experiments were carried out at three distinct samples