Figure 4. The antagonistic crosstalk between Ser 140 phosphorylation and Arg 138 methylation of p16 protein.

(A) 293T cells were transfected with empty control vector (pWPXLD), wild type p16, or mutant p16 expression plasmids. After 24 h the cells were treated with 1 mM H₂O₂ for 30 min, and after 24 h the apoptosis was evaluated by flow cytometry. Apoptosis rates were calculated on the basis of 15 000 cells. (B,C) CoIP assays with anti-GFP or anti-p16 and detected with anti-p16, anti-ASYM or anti-phosphserine antibody. 293T cells transfected with wild type p16 or mutant p16 expression plasmids. After 24 h the cells were treated with 1 mM H₂O₂ for 30 min, and cultured 24 h before harvest. Upper: The western of p16, mehtylated-p16, phosphorylated-p16 and actin as indicated. Lower: The results were the photodensitometry analysis of the western bands from three experiments, and the results are presented as the relative intensity ratio between phosphorylation or methylation bands and IP-p16 bands.