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

Figure 3. Caveolin-1-mediated endocytosis of p53 core domain.

Figure 3

(A) A549 cells, which were transfected with siRNA-targeting caveolin-1, were treated with RP53 for 2 h in a dose-dependent manner. Western blot analysis was performed with the indicated antibodies. Actin was used as a loading control. (B) Blocking of Ras activity or complete knock-down of caveolin-1 suppressed the endocytosis of RP53. HCT116 cells which were transfected with DN-Ras (Dominant Negative) and siRNA-targeting caveolin-1 were treated with RP53 for 2 h in a dose-dependent manner. Western blot analysis was performed with the indicated antibodies. Actin was used as a loading control. (C) Internalized RP53 was co-localized with caveolin-1 in time-dependent manner. HCT116 was treated with FITC or FITC-p53 following indicated time. After MeOH fixation, cells were stained with caveiolin-1 antibody (Red) and analyzed by confocal microscopy. Green (FITC) indicated endocytosed RP53. Inset indicated enlarged figure of boxed region. (D) EEA1, an early endosome marker, was also co-localized with treated RP53 in time dependent manner. Immunofluorence (IF) staining was performed with the protocol above, and the images were obtained using confocal microscopy. Inset indicated enlarged figures of boxed region. (E) Determination of key region for endocytosis through fragment mapping. The p53 DNA-binding domain (93-292 AA) was cleaved into 4 small fragments with 50 AA each, which were then tagged with GST. (F) Among the fragments, p53 #1 (93-142 AA) was specifically internalized into K-Ras mutated cell lines (A549 and HCT116). 4 cancer cell lines were incubated with either RP53 or with the 4 different fragments of p53. After 2 h, cells were washed twice with PBS and analyzed by western blot using the indicated antibodies. Actin was used as a loading control. (G) p53 #1 directly interacted to caveoin-1. Agarose bead- conjugated GST-p53 (DNA binding Domain; 93-292AA) and p53 fragments (p53 #1-4) were incubated with A549 cell lysates. GST-pull down was performed to monitor p53-associated proteins.