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. Author manuscript; available in PMC: 2009 Aug 24.
Published in final edited form as: Mol Cell. 2008 Oct 24;32(2):180–189. doi: 10.1016/j.molcel.2008.08.031

Figure 3. Mdm2 promotes the proteasomal degradation of polyubiquitylated L26.

Figure 3

U2OS cells were seeded at 8×105 cells/10cm dish. Twenty four hours later, cultures were transfected with expression plasmids encoding L26-Flag (3μg/dish), human wtMdm2 (wt, 2.5μg/dish), Mdm2ΔR (ΔR, 2.5μg/dish), HA-ubiquitin (1μg/dish), and GFP (0.25μg/dish) as internal transfection control. After one day, cells were treated with 8μM MG132 overnight, harvested and extracted in SDS lysis buffer under denaturing conditions. WCE: 2.5% of the extract was resolved by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies. IP: the rest of the extract was immunoprecipitated with anti-Flag antibodies, and subjected to SDS-PAGE followed by Western blot analysis with anti-HA antibodies (IB: HA).