Fig. 2: β-adaptin binding is required for p53 and mutant p53 packing into the exosomes.

A Co-immunoprecipitation (Co-IP) analysis of the cytoplasm fraction of the endogenous p53^R273H-expressing HT-29 and endogenous p53^R175H-expressing KLE cells, with Lamin B1 and α-tubulin considered as the markers of nuclear and cytoplasm fractions, respectively. Anti-p53(sc-126) were used to immunoprecipitated and Anti-p53 (#2527) and anti-β-adaptin (sc-74423) were used for WB. B Representative images of immunofluorescence stained HEK293 cells showing endogenous β-adaptin (green), exogenous FLAG-p53^WT and FLG-p53^R273H (red), and DAPI for nuclei (blue; scale bar: 10 μm). C Co-IP analysis of Flag-p53^WT or p53^LL25AA mutants with endogenous β-adaptin in HEK293 cells. The whole cell lysates were extracted and immunoprecipitated using anti-β-adaptin. D Expression of p53^R273H in the exosomes isolated from the H1299 stable expressed p53^R273H cell culture supernatant after β1-adaptin knockdown (si-AP1B1). Alix, tsg101, and CD63 were considered as the positive markers, while calnexin was considered as a negative marker for exosomes. Anti-AP1B1 (16932-I-AP) was used to detect the silence efficiency. E Expression of p53^R273H in the exosomes isolated from the H1299 cell culture supernatant after co-transfection with exogenous GFP-p53^R273H and/or FLAG-AP1B1 cell culture supernatants. Alix, tsg101, and CD63 were considered as the positive markers, while calnexin was considered as a negative marker for exosomes.