A. U2OS cells were seeded at 800,000 cells/10cm dish. Twenty four hours later, cells were transfected with expression plasmids encoding L26-Flag (6μg/dish), human wtMdm2 (wt, 3μg/dish), Mdm2ΔR (ΔR, 3μg/dish), HA-ubiquitin (1.5μg/dish), and GFP (0.4μg/dish) as transfection control. After two days, cells were treated with 25μM MG132 for 4 hours, harvested and extracted in SDS lysis buffer under denaturing conditions. WCE: 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). The membrane was re-blotted with anti-Flag antibodies to detect immunoprecipitated non-ubiquitylated L26 (bottom panel). Positions of molecular size markers are indicated on the right.
B. U2OS cells (2×10⁶ cells/15cm dish) were co-transfected with expression plasmids encoding L26-Flag (6.5μg/dish), Mdm2 (3.2μg/dish), HA-ubiquitin (2.7μg/dish), and GFP (0.5μg/dish) as transfection control. One day later, cells were trypsinized and separated into nuclear and cytoplasmic fractions. Extract: 4% of each fraction was resolved by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies. NUC: nucleus; CYTO: cytoplasm. IP: the rest of each extract was subjected to denaturing conditions (SDS lysis buffer), immunoprecipitated with anti-Flag antibodies, and subjected to SDS-PAGE followed by Western blot analysis with anti-HA antibodies and reblotting with anti-Flag antibodies. Ub-L26-Flag: ubiquitylated L26-Flag. Positions of molecular size markers are indicated on the right.

A. Transfected Mdm2 augments endogenous L26 polyubiquitylation. U2OS cells were transfected with the indicated combinations of Mdm2 and HA-ubiquitin (HA-Ub) expression plasmids. Cells were extracted in SDS lysis buffer under denaturing conditions and subjected to immunoprecipitation (IP) with either L26-specific antibodies or anti-FLAG rabbit polyclonal antibodies as control, followed by Western blot analysis with antibodies directed against HA to detect ubiquitin conjugates. Arrows indicate bands migrating at the positions expected for monoubiquitylated and diubiquitylated L26. Upper panels: analysis of whole cell extracts (WCE) with the indicated antibodies. Ub-p53 indicates the positions of monoubiquitylated and diubiquitylated p53. GAPDH was used as loading control.
B. Knockdown of endogenous Mdm2 reduces the polyubiquitylation of transfected L26. MCF-7 cells were transfected with the indicated expression plasmid combinations, along with synthetic Mdm2-specific siRNA oligonucleotides or control siRNA. Where indicated, MG132 treatment was performed prior to cell harvesting. Following extraction in urea lysis buffer (see Experimental Procedures), lysates were precipitated with Ni-NTA magnetic agarose beads (Qiagen) and the immunoprecipitated material was resolved by SDS-PAGE and analyzed by Western blot analysis with anti-HA antibodies to visualize the precipitated ubiquitylated L26 (bottom panel, Pull-down). Upper panels: analysis of whole cell extracts (WCE).
C. Knockdown of endogenous Mdm2 reduces the polyubiquitylation of endogenous L26. U2OS cells were seeded at 1.25×10⁶ cells/15cm dish. Twenty four hours later, cells were transfected with siRNA (SMART pool, Dharmacon) directed against Mdm2, or control siRNA (SMART control). After one day, cultures were transfected with plasmids encodig HA-ubiquitin (2.5μg/dish) and GFP. The next evening, cultures were treated with 10μM MG132 overnight. Cells were then harvested and extracted in SDS lysis buffer under denaturing conditions. WCE (upper panels): 1.8% of the extract was resolved by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies. GFP was employed as a transfection efficiency control. IP (lower panels): Extracts were subjected to immunoprecipitation with anti-L26 antibodies followed by Western blot analysis with antibodies directed against HA to detect ubiquitin conjugates. The positions of total ubiquitylated L26 (Ub-L26) and of polyubiquitylated L26 are indicated. A short (SE) and long (LE) exposure of the same membrane are shown. The membrane was re-blotted with anti-L26 antibodies to quantify the total immunoprecipitated L26 protein (bottom panel, very short exposure shown). To calculate the relative extent of endogenous L26 polyubiquitylation, (Poly Ub-L26/relative), the polyubiquitination signal of L26 (polyUb-L26, short exposure) was quantified using ImageJ software, divided by the corresponding GFP signal and normalized to 1.0.

PCR products consisting of a T7 promoter-tagged p53 cDNA harboring a 1nt or 110nt 5′UTR, L26 cDNA and human Mdm2 cDNA (wild type (wt), RING domain deletion (ΔR), acidic domain deletion (ΔA)) were in vitro transcribed/translated in rabbit reticulocyte lysates in the presence of [³⁵S] methionine. Reticulocyte lysates were pre-treated with 50μM MG132 for 15 minutes at room temperature prior to the addition of PCR-generated cDNAs. Lysates were resolved by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies.
A. Effect of L26 on translation of p53 mRNA harboring a 1 or 110nt 5′UTR in the absence or presence of increasing wtMdm2 (1:1 and 1:2 ratio of L26 cDNA to Mdm2 cDNA). Amounts of total L26, including the endogenous L26 of the reticulocyte lysate, is shown in the middle panel. Equal loading was assessed by Ponceau staining of the membrane (bottom panel).
B. Effect of L26 on translation of p53 mRNA harboring a 110nt 5′UTR in the absence or presence of increasing Mdm2 (wild-type, ΔR, ΔA). Analysis was as in A.

A. HEK293 cells (1.75×10⁶ cells/10cm dish) were transiently co-transfected with the indicated combinations of expression plasmids encoding L26-Flag (6μg/dish), wild type human Mdm2 (6μg/dish), and GFP (0.3μg/dish) as a transfection control. Cells were harvested and extracted 48 hours later. Top panel: Extracts were immunoprecipitated (IP) with anti-Flag or anti-Mdm2 antibodies, and the immunoprecipitated material was resolved by SDS-PAGE and subjected to Western immunoblot analysis (IB) with the indicated antibodies. LC: antibody light chain. Bottom panel: 2.5% of each whole cell extract (WCE) was resolved by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies.
B. SJSA-1 cell cultures were treated with 8μM MG132 overnight where indicated, or left untreated. The next day, cells were harvested and subjected to protein extractions. Extracts were immunoprecipitated (IP) with control (anti-Flag rabbit polyclonal, upper panel, lane 1) or anti-L26 (lanes 2, 3) antibody (two 15cm dishes/lane). For the reciprocal reaction, extracts were immunoprecipitated with control (anti-SV40 large T antigen, lane 4) or anti-Mdm2 (lanes 5, 6) antibodies (four 15cm dishes/lane). The immunoprecipitated material was resolved by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies (IB). Lower panel: 1.7% of each whole cell extract was analyzed directly by SDS-PAGE, followed by Western blot analysis (IB) with the indicated antibodies.

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).

MCF7 cells (3×10⁶ cells/10cm dish) were co-transfected with expression plasmids encoding GFP (4μg/dish); lanes 1,5) or GFP-L26 (4μg/dish); lanes 2–4, 6–8), without or with increasing amounts of human Mdm2 (4μg or 8μg/dish) expression plasmid. The total amount of DNA was kept at 12μg by the inclusion of plain pCMV-Neo-Bam vector. Forty-eight hours post-transfection, cultures were exposed to ionizing radiation (IR; 10Gy) as indicated and harvested 30 minutes post-IR. Lysates were immunoprecipitated with anti-GFP antibodies and the amount of coprecipitated p53 mRNA was assessed by IP-RT-PCR, employing primers that map within the 5′-UTR of p53 mRNA and span an intron within the genomic human p53 DNA. PCR products were visualized by ethidium bromide staining (top panel). Protein input for each IP reaction was resolved by SDS-PAGE and assessed by Western blot analysis with the indicated antibodies (bottom panels).

A. MCF7 cells were transiently transfected with the corresponding Mdm2 expression plasmids and treated with 50μM MG132 for 30 minutes prior to exposure to 10Gy ionizing radiation (IR). Twenty five minutes after IR the cells were pulse-labeled for 5 minutes with [³⁵S]methionine. p53 protein was immunoprecipitated (IP) and the level of immunoprecipitated radiolabeled protein was assessed by autoradiography (³⁵S); Western blot analysis was employed to assess the total amount of immunoprecipitated p53 (IB:p53). Autoradiography of hole cell extracts (WCE, ³⁵S, middle panel) confirm equal amounts of overall [³⁵S]methionine incorporation into cellular proteins and Western blot analysis of WCE shows total levels of Mdm2 and p53. Numbers below upper panel indicate the relative increase in p53 synthesis upon IR treatment for each pair of samples.
B. Model depicting the involvement of L26 in the p53-Mdm2 autoregulatory loop. Transcription of the mdm2 gene is positively regulated by p53. In turn, the Mdm2 protein binds p53, inhibits its biochemical activities and targets it for ubiquitin-dependent proteasomal degradation. Translation of p53 mRNA is positively regulated by L26, but Mdm2 reduces this effect by promoting L26 degradation as well as interfering with the binding of L26 to p53 mRNA. Thus, Mdm2 downregulates p53 by both inhibiting its synthesis and augmenting its demise.