Figure 3.

EZH2 cannot methylate Ku80. (a) Purified CD4⁺and CD8⁺T cells isolated from PBMC were stimulated with anti-CD3/CD28 antibodies plus IL-2. Cells were collected at different time points for immunoblotting. The band densitometry was quantified using ImageJ software. The quantitative data were calculated from three independent experiments, and were shown the mean±S.E.M. *P<0.05. (b) CD4⁺and CD8⁺T cells were stimulated with anti-CD3/CD28 antibodies plus IL-2 for 72 h and then collected for immunostaining analysis. (c and d) CD4⁺and CD8⁺T cells were stimulated with anti-CD3/CD28 antibodies plus IL-2 for 72 h. Cells were transfected by electroporation with Ku80 and EZH2 siRNA or control siRNA and harvested after 48 h. The cell lysates were subjected to immunoblotting. (e) 293 T cells transfected with plasmids encoding Flag-Ku80 and SET domain-truncated EZH2 (ΔSET) with Myc-tag were immunoprecipitated with anti-Flag antibody, and the precipitates were subjected to immunoblotting. (f) Jurkat T cells were transfected by electroporation with EZH2 siRNA or control siRNA and harvested after 48 h. The cells were immunoprecipitated with anti-Ku80 antibody, and the precipitates were subjected to immunoblotting. *Represents a non-specific band at~80 kDa