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. 2010 Jan 19;120(2):521–532. doi: 10.1172/JCI40706

Figure 3. NNK enhances DNMT1 protein stability through the AKT signaling pathway, which is associated with the ubiquitin-proteosome system.

Figure 3

(A) NNK prolonged DNMT1 protein half-life. DNMT1 protein levels were determined by Western blotting and densitometry. β-Actin was used as a loading control, and p53 served as a positive control for CHX treatment because its half-life is known to be short. White bars, cells treated with DMSO; dark gray bars, cells treated with CHX for 6 hours; light gray bars, cells treated with CHX and NNK for the indicated times. (B) Ubiquitination of DNMT1 decreased upon NNK treatment. A549 cells were treated with NNK or DMSO control for 2 hours, after which cell lysates were immunoprecipitated with anti-DNMT1 or anti-ubiquitin antibody and then Western blotted. Normal IgG was used as a negative control. (C and D) A549 cells were pretreated with or without the PI3K/AKT pathway inhibitor LY294002 (C) or AKT siRNA (D), then treated with NNK. Western blotting and densitometry showed that each reduced levels of NNK-induced DNMT1. (E) LY294002 treatment increased ubiquitination of DNMT1, leading to low levels of DNMT1 protein in A549 cells. (F) Western blotting and densitometry of A549 cells treated with NNK with or without pretreatment with LY294002 and the proteasome inhibitor MG132 showed altered DNMT1 protein levels. Data are mean ± SEM (n = 3).