Figure 5.

Mapping of the 32 kDa band C-terminal region. (A and B) H1299 cells were transfected with an expression vector for hMdm2 (pCMVhMdm2) and left untreated (−) or treated with 2 nM LMB for 18 h (+). Cell extracts were analysed by Western blotting and hMdm2 was detected using the mouse monoclonal antibodies 2A9, 4B2, SMP14 and 2A9 (A), or 4B2, 4B11 and 2A10. (B). The positions of the bands corresponding to the full-length hMdm2 and the 32 kDa fragment are indicated by arrows. (C) H1299 cells were transfected with an expression vector for hMdm2 (pCMVhMdm2) (lane1), a vector expressing residues 1–244 of murine Mdm2 (pcDNA3Mdm2 1–244) or a vector expressing residues 1–258 (pcDNA3Mdm2 1–258) (lane3). pcDNA3Mdm2 1–244 and pcDNA3Mdm2 1–258 contain six and 14 additional residues at their C-terminus, respectively. (D) H1299 cells were transfected with expression vectors for hMdm2(1–252N₄₇) (lanes 1 and 2) or hMdm2 (lanes 3 and 4). In lanes 2 and 3, cells were treated with 2 nM LMB for 18 h. Cell extracts were analysed by Western blotting and developed with the 4B2 antibody. The position of the 32 kDa band is indicated with an arrow. In lane 5, cells were transfected with the plasmid encoding the hMdm2 (1–244) mutant. This fragment is expressed at very high levels, and therefore, a shorter exposure of the gel is shown for this lane. (E) Sequence of the acidic domain in hMdm2. The regions involved in the interaction with the amino-terminus of p14ARF according to Bothner et al (2001) and the 2A10 epitope are underlined. The region proposed to contain the C-terminus of the 32 kDa band is marked with a discontinuous line.