Journal: Cell Cycle

Article Title: The alternative translated MDMX p60 isoform regulates MDM2 activity

doi: 10.4161/15384101.2014.977081

Figure Lengend Snippet: hMDMX p60 shows higher affinity toward hMDM2 as compared to hMDMX FL . ( A ) Immunoprecipitation (IP) against hMDM2 shows that hMDM2 interacts with both hMDMX isoforms (upper panel). The protein levels in whole lysates are shown below. (NS stands for non-specific). ( B ) Co-immunoprecipitation of hMDMX isoforms (left) or hMDM2 (right) using an HA-tagged hMDMX p60 . Hsp90 and actin serves as loading controls of total lysates. ( C ) ELISA using a fixed (500 ng) amount of recombinant purified hMDMX p60 or hMDMX FL and increasing amounts of hMDM2. The affinity of hMDMX p60 to hMDM2 is higher as compared to hMDMX FL . Both proteins bind hMDM2 in a biphasic fashion. The data shows the average of 7 independent experiments and SD. Western blots ( A and B ) show one representative experiment out of 3.

Article Snippet: In vitro ubiquitination assays were performed 2 h at 37°C, using reaction mix containing: E3 hMDM2 recombinant protein (amount as indicated on the figure) and an equal amount of Ubiquitin-Activating Enzyme E1, (VWR); Ubiquitin Conjugating Enzyme 5 b (VWR) and in presence or not of Ubiquitin His-Tagged (VWR) in a solution containing 1 μM ATP (Invitrogen); 1 μM DTT (Invitrogen).

Techniques: Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Recombinant, Purification, Western Blot

Journal: Cell Cycle

Article Title: The alternative translated MDMX p60 isoform regulates MDM2 activity

doi: 10.4161/15384101.2014.977081

Figure Lengend Snippet: hMDMX p60 stabilizes hMDMX FL in the presence of hMDM2. ( A ) The levels of expression of hMDMX FL in H1299 cells following increasing amounts of hMDM2 (left). A similar experiment but using a fixed amount of hMDMX p60 and increasing levels of hMDM2 (right). Compare differences in the expression of hMDMX isoform in cells transfected with 50 ng hmdm2 cDNA. ( B ) hMDMX FL is protected from hMDM2-mediated degradation in the presence of hMDMX p60 . ( C ) Increasing levels of hMDMX p60 stabilizes hMDMXFL in cells expressing endogenous hMDM2. ( D ) Mutation of each, or all 6 together, of the lysine residues in the N-terminus of hMDMX upstream of the initiation site for hMDMX p60 do not affect hMDM2-mediated degradation of hMDMX. The data are representative from 3 independent experiments ( Fig. S3A and B ).

Article Snippet: In vitro ubiquitination assays were performed 2 h at 37°C, using reaction mix containing: E3 hMDM2 recombinant protein (amount as indicated on the figure) and an equal amount of Ubiquitin-Activating Enzyme E1, (VWR); Ubiquitin Conjugating Enzyme 5 b (VWR) and in presence or not of Ubiquitin His-Tagged (VWR) in a solution containing 1 μM ATP (Invitrogen); 1 μM DTT (Invitrogen).

Techniques: Expressing, Transfection, Mutagenesis

Journal: Cell Cycle

Article Title: The alternative translated MDMX p60 isoform regulates MDM2 activity

doi: 10.4161/15384101.2014.977081

Figure Lengend Snippet: Increasing levels of hMDMX p60 induces oscillation in hMDM2 expression levels in H1299 cells. ( A ) Expression of a fixed amount of hmdm2 and increasing levels of either (a) hMDMX p60 alone or (b) together with a fixed amount of hmdmx fl , or (c) increasing levels of both isoforms. The hmdm2( C464A ) mutant (d) is E3 ligase dead and is not affected by hMDMX p60 ( Fig. S4A and B ). ( B ) In vitro autoubiquitination of recombinant hMDM2 in the presence of increasing levels of hMDMX p60 . Data shows one representative experiment out of 3 ( Fig. S5A and B ).

Article Snippet: In vitro ubiquitination assays were performed 2 h at 37°C, using reaction mix containing: E3 hMDM2 recombinant protein (amount as indicated on the figure) and an equal amount of Ubiquitin-Activating Enzyme E1, (VWR); Ubiquitin Conjugating Enzyme 5 b (VWR) and in presence or not of Ubiquitin His-Tagged (VWR) in a solution containing 1 μM ATP (Invitrogen); 1 μM DTT (Invitrogen).

Techniques: Expressing, Mutagenesis, In Vitro, Recombinant