Figure 2.

LMB induces the appearance of novel forms of hMdm2. (A) H1299 cells were transfected with an expression vector for hMdm2 (pCMVhMdm2) and left untreated (lane 1) or treated with 2 nM LMB for 18 h (lane 2). Cell extracts were analysed by Western blotting and hMdm2 was detected using the 4B2 mouse monoclonal antibody against the amino-terminus of the protein. The positions of the bands corresponding to the full-length hMdm2, the LMB-induced slower mobility form and the 32 kDa fragment are indicated by arrows. (B) LMB does not affect hMdm2 ubiquitination in vivo. H1299 cells were transfected with 5 μg pCMVhMdm2 and 2 μg pCMVHis₆–Ubiquitin. In lanes 1 and 3, cells were left untreated, in lanes 2 and 4 they were treated with 2 nM LMB and in lanes 3 and 6, they were also cotransfected with 5 μg of the pcDNA3p14ARF expression vector. In lanes 1–3, total hMdm2 was analysed and in lanes 4–6, His₆–ubiquitin complexes were isolated and ubiquitinated hMdm2 was detected using 4B2.