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. 2013 Dec 2;4(12):2523–2531. doi: 10.18632/oncotarget.1550

Figure 2. Active internalization of p53 core domain occurs by K-Ras dependent manner.

Figure 2

(A-B) K-Ras dependent endocytosis was a p53-specific event. RP53 and recombinant Snail (Snail middle region; GST tag; 91-121 amino acids (AA)) were treated in a time-dependent manner for up to 12h in the K-Ras harboring A549 cells. The endocytosis levels were measured by western blot analysis using the indicated antibodies. (C) Internalization of RP53 was appeared through active mechanism. Cells were divided into two groups; one group was incubated with FITC-p53 first and then fixed with 2% PFA, while the other group was exposed to PFA before FITC-p53 treatment. Both groups were stained with PI (propidium iodide; Red) for nuclear staining. (D) Dynamin inhibitor remarkably reduced endocytosis of RP53 in HCT116 cells. After incubation with Dynasore (from 20 to 80 μM) for 3 h, cells were treated with FITC or FITC-p53 for 1 h as an indicated dose. After washing with PBS, cells were harvested and subjected to SDS-PAGE. Western blot analysis was performed with indicated antibodies. (E) Decreased internalization of RP53 in A549 was measured by FACS analysis. (F) Internalized RP53 was quantified by graph. Numbers of Y-axis showed the value of FITC-p53 positive portion/FITC positive portion. After incubation with Dynasore (80 μM) for 3 h, cells were treated with FITC or FITC-p53 (2 μg/ml) for 1 h.