MA1-113

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MDM2 was performed by loading 20 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a Novex 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. MDM2 was detected at ~57, ~75, and ~90 kDa using a MDM2 monoclonal antibody (Product # MA1-113) at a dilution of 1 µg/mL in 5% Milk in TBST overnight at 4C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A28177) at a dilution of 1:1000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal Pico substrate (Product # 34078) and the myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MDM2 was performed with 10 µg of A549 cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against MDM2 (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. MDM2 was detected at ~ 74 kDa using MDM2 Mouse monoclonal antibody (Product # MA1-113) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Mouse (Product # 31437) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). MDM2 Antibody (Product # MA1-113) confirms silencing of MDM2 expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of MDM2 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of monoclonal MDM2 antibody (Product # MA1-113) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of MDM2 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of monoclonal MDM2 antibody (Product # MA1-113) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. MDM2 protein expression in cutaneous melanoma. Representative images of (A) weak MDM2 immunostaining in pT1 melanoma and (B) in pT2 melanoma, (C) moderate MDM2 immunostaining in pT3 melanoma and (D) weak MDM2immunostaining in pT4 melanoma. Marked areas indicate MDM2 staining in malignant melanocytes. Magnification, x100. pT, pathological tumor stage.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: EV4 Figure Evaluation of PIDDosome expression levels and its prognostic value in human HCC Densitometric quantification of the immunoblot in Fig 5A is shown as the fold change in tumor (T) tissue over non-tumorous (NT) tissue of the individual patients. The values for proCASP2, PIDD-C, and RAIDD were normalized to the corresponding PonceauS staining. Immunoblot of the HCC cell line SNU182 treated with DMSO or increasing concentrations of the Aurora B inhibitor ZM447439. The membrane was probed for CASP2, RAIDD, MDM2, p53, and HSP90 as loading control. Recurrence-free survival of HCC patients in the TGCA Provisional data set (LIHC) expressing high or low levels of the proliferation-associated genes Ki67 , E2F1, and CCNA2 . Disease-free survival of the TGCA HCC patients shown in Fig 5C was analyzed with respect to the p53 (mutated or wt) status. Data are divided at the median, and statistical significance was tested using a log-rank test. Butterfly plots showing the enrichment of E2F target genes in the co-expression networks of CASP2 , RAIDD, and PIDD1 in the same TCGA data set. Source data are available online for this figure.