Fig. 3: CHK2-mediated phosphorylation of mutant p53 Ser20 inhibits secretion.

Expression of endogenous p53^R273H in the exosomes isolated from the HT-29 (p53^R273H) cell culture supernatant after treatment without or with post-10Gy IR for 4 h (A) and 500 nM DOX for 12 h (B). Alix, tsg101, CD9, CD81 and CD63 were considered as the positive markers, while calnexin was considered as a negative marker for exosomes. C Expression of exogenous p53 mutations immunoprecipitated by anti-FLAG from the HEK293 cell culture supernatant after transfection with FLAG-p53^WT, FLAG-p53^S20E and FLAG-p53^S20A. Expression of exogenous p53^R273H immunoprecipitated by anti-FLAG or anti-GFP from the HEK293 cell culture supernatant after transfection with FLAG-p53^R273H/GFP-p53^R273H cell culture supernatants after knocking-down CHK2 (D) or overexpression of FLAG-CHK2 (E). F Co-IP analysis of FLAG-p53^WT, FLAG-p53^S20E and FLAG-p53^S20A mutants with endogenous β-adaptin in HEK293 cells. The whole cell lysates were extracted and immunoprecipitated using anti-FLAG. G Expression of endogenous p53 and β-adaptin using anti-p53 to perform Co-IP assay in HT-29 (p53^R273H) cell after treatment without or with 10 Gy IR (left) and 500 nM DOX (right). Expression of p53 and β-adaptin using anti-GFP to perform Co-IP assay in HEK293 cells after transfection with GFP-p53^R273H and knocking-down of CHK2 (H) or overexpression of FLAG-CHK2 (I).