FIG 5.
HDM2(S395D) and HDMXS(403D) interact through the N-terminal domain of both proteins. (a) Cartoon illustrating the constructs HDM2(1-301) and HDMX(1-255). Recombinant purified HDMX(1-255) is shown. (b) PLA using anti-HDMX and anti-HDM2. H1299 cells were transfected with the indicated constructs. Cell nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) (blue). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (c) Constructs of HDMX and HMD2 lacking the Zn finger and the RING domain were tested by ELISA. The Kd (46.9 nM) of the interaction is similar to the one calculated for the interaction between the phosphomimetic mutants. (d) The N-terminal HDMX domain was tested for its ability to recognize different HDM2 domains. In a pDUET plasmid we cloned the N terminus of HDM2(1-100) with a His tag and the acidic domain of HDM2(241-335) without any tag. The total lysate of pDUET construct was pulled down with GST-HDMX(1-110) to observe if the N terminus of HDMX interacts with one of the two domains of HDM2 cloned in pDUET. The membrane was probed with anti-HDM2 4B2 (epitope 29-50), showing the N terminus of HDM2, and anti-His, showing both N-termini, because GST-HDMX(1-110) also has a His tag, and 2A10 (epitope 255-262), which did not react because the acidic domain of HDM2 does not interact with GST-HDMX(1-110).