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. 2005 Dec 23;393(Pt 2):489–501. doi: 10.1042/BJ20050960

Figure 4. LZAP restores HDM2 ubiquitin E3 ligase activity towards p53 in the presence of ARF.

Figure 4

(A) U2OS cells were transfected with combinations of plasmids as indicated and, 4 h before lysis, treated with the proteasome inhibitor MG132. Lysates were immunoprecipitated with 0.6 μg of goat anti-p53 antibody (α-p53) (FL393), and HA–ubiquitin-modified p53 was visualized using a HRP-conjugated mouse anti-HA antibody (α-HA) (HRP-F7). (B) U2OS cells were transfected as indicated. Immunocomplexes immobilized on Protein G–beads were prepared using mouse anti-HDM2 antibody (α-HDM2) (SMP14, lanes 1–5) or anti-ARF serum (α-ARF) (10437, lanes 6 and 7), followed by an ubiquitination reaction. Identical lysates were immunoprecipitated (IP) in lanes 3 and 6, as well as lanes 4 and 7. Top panel: ubiquitinated p53 ladders were detected using goat anti-p53 antibody (α-p53) (FL393). Middle panel: the presence of HDM2 was confirmed by immunoblotting with SMP14. Bottom panels: for both in vivo and in vitro assays, lysates were separated by SDS/15% PAGE and immunoblotted with antibodies against the indicated proteins as a marker of protein expression (bottom panels). Ub, ubiquitin.