33-7100

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MDM2 was performed by loading 30 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 # 33-7100) at a dilution of 2.5 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 by loading 30 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 # 33-7100) at a dilution of 2.5 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: Knockdown of MDM2 was achieved by transfecting U-2 OS with MDM2 specific siRNAs (Silencer® select Product # S8628, S8629). Western blot analysis (Fig. a) was performed using Whole cell extracts from the MDM2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with MDM2 Monoclonal Antibody (IF2) (Product # 33-7100, 1 µg/mL ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knockdown confirms that antibody is specific to MDM2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-MDM2 Monoclonal Antibody (IF2) (Product # 33-7100) and a 75 kDa band along with 55 kDa band, both corresponding to MDM2 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of U-2 OS (Lane 1) and A549 (Lane 2) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. (a) Hematoxylin and eosin stain (40x magnification) from the first, atypicalmeningioma showed a highly cellular tumor with large nuclei and prominentnucleoli, and an increased mitotic count. (b) The tumor showed a varyingexpression pattern for EMA (20x magnification). (c) SSTR2A showed diffuseand strong membranous and cytoplasmic positivity (20x magnification). (d)MDM2 amplification resulted in MDM2 overexpression, since additionalimmunohistochemistry showed nuclear positivity for MDM2 (40xmagnification).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. a) Hematoxylin and eosin stain (40x magnification) from the recurrent,anaplastic meningioma showed a frankly malignant tumor with prominentcytological atypia, high mitotic count, and extensive necrosis. (b) Thetumor showed only partial positivity for EMA (20x magnification). (c) SSTR2Ashowed membranous and cytoplasmic positivity in the majority of the tumor(40x magnification). (d) MDM2 amplification resulted in MDM2 overexpression,since additional immunohistochemistry showed nuclear positivity for MDM2(40x magnification).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 P-STAT5 regulates p53/MDM2 functions and cell survival through NPM1 protein. ( a ) HEK 293T cells (6 x 10 5 per well) transfected with either RFP-STAT5A or RFP-backbone vectors were harvested at 24 h after transfection, and the cell lysates were subjected to immunoblotting assays for detection of expression levels of p53 and its phosphorylation levels at serine 15 and threonine 18, as well as the expression levels of MDM2 and its phosphorylation level at serine 166. ( b ) Six hours after transfection with either RFP-STAT5A or RFP-backbone vectors, HEK 293T cells (6 x 10 5 per well) were incubated overnight with inhibitor 573108 at a concentration of 340 mu M and then harvested and lysed for immunoblotting assays to examine the expression levels of p53 and MDM2 as well as their relevant phosphorylation levels. ( c and d ) HEK 293T cells (6 x 10 5 per well) were transfected with RFP-STAT5AY694F mutant vector for 24 h, harvested and lysed for immunoblotting assays on the expression levels of MDM2, p53 and p21. ( e ) HEK 293T cells (6 x 10 5 per well) were co-transfected with RFP-wtSTAT5A and FLAG-NPM1 vectors for 24 h, harvested and lysed for immunoblotting assays on the p53 expression levels. ( f ) MCF-7 cells (2 x 10 6 per well) were plated on 10 cm plate on day 0 and then lentivirally transduced with pLVX-IRES-mCherry-wtSTAT5 or pLVX-IRES-mCherry-STAT5Y694F on day 1. The cells were harvested 72 h after the transduction and lysed for immunoblotting assays. ( g ) HEK 2

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 USP2 is induced by PUVA and a p53 activator, nutlin3a p53 wt CTCL cell line, MyLa2000, was subjected to PUVA a, b. or 5muM nutlin3a c-d. as shown in Methods. USP2 expression was measured by qPCR (a, c) and western blot (WB) (b, d) 2h-72h after the treatments. For qPCR, data was normalized to GAPDH and expressed as relative units (RU). The experiments were repeated 3 times, unpaired T-test was used to calculate P-value, error-bars, SD, *, P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 USP2 inhibits the cellular actions of p53 through Mdm2 a. USP2 knockdown decreased Mdm2 expression. MyLa2000 cells were transfected with either siRNA control or USP2 siRNA for 24h, followed by treatment with 5muM nutlin3a for 24h. Western blot was used for detecting USP2, Mdm2 and p53 protein level. beta-actin was used as internal control, and relative protein expression levels are reported below the corresponding western blot bands. The experiment was repeated 3 times. Representative data was shown. b. USP2 knockdown increase p53 transcription activity. The expression of p21 was used to indicate p53 transcription activity. qPCR was applied to examine p21 expression in MyLa2000 cells transfected with siUSP2/siRNA with or without nutlin3a treatment. Paired T-test was used to calculate P-value. c. A proposed model for the regulation of USP2. PUVA and nutlin3a activate p53 and promote cell apoptosis. USP2 is induced by PUVA and nutlin3a and increases cell resistance to apoptosis via modulation of Mdm2/p53 interaction, constituting an anti-apoptosis protective loop.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 RITA restores TP53 activity in medulloblastoma cell lines with and without TP53 mutations ( A ) TP53 and CDKN1A protein expression after 48 h of RITA treatment assessed by western blot. Ethanol served as a negative control. wt = wildtype, mut = mutation. ( B, C ) Relative expression of CDKN1A and MDM2 mRNA after 48 h of RITA treatment measured by real-time polymerase chain reaction (PCR). Ethanol served as a negative control. * = P < 0.05 in Student's t-test .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 CDH17 activates multiple signal transduction pathways. A. Expression of beta-catenin, GSK-3beta, p- GSK-3beta, Rb, Cyclin D1were compared between shCDH17 and Mock both in AGS and MKN-45 cells by Western blot analysis. MDM2, p53 and p21 also were evaluated. beta-Actin was used as a loading control. B. TOPflash/FOPflash reporter assay shows that Wnt signaling re-activated after CDH17 restoration in AGS and MKN-45. (* P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 Representative cases of IHC expression (magnification 400X). Cases of IHC expression with (A) MDM2+, (B) MDM2-, (C) p53+, and (D) p53- were demonstrated. The positive staining was predominantly nuclear for both MDM2 and p53.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure S5. Transcriptome-wide RNA-seq, m 6 A-seq, and RIP-seq assays in murine NK cells. (A) Volcano plots of differentially expressed genes from RNA-seq. (B) GSEA showing enrichment of E2F targets, G2/M checkpoint, and mitotic spindle hallmark gene sets in Ythdf2 DeltaNK NK cells compared with Ythdf2 WT NK cells. (C) The proportion of m 6 A peak distribution in NK cells from Ythdf2 WT and Ythdf2 DeltaNK mice. (D) GO analysis of transcripts with m 6 A peaks. (E) Density distribution of the YTHDF2-binding sites across mRNA transcriptome from RIP-seq data. (F) The proportion of YTHDF2 binding site distribution from RIP-seq data. (G) Top 10 GO clusters from GO analysis of YTHDF2 target genes from RIP-seq data. (H) Immunoblotting showing the protein levels of MDM2, TDP-43, and YTHDF2 in IL-2-expanded NK cells enriched from the spleen of Ythdf2 WT and Ythdf2 DeltaNK mice. (I and J) qPCR and immunoblotting showing the expression of Mdm2 (I) and Tardbp (J) in NK cells transfected with gene-specific or control siRNA. (K and L) IL-2-expanded NK cells were transfected with Mdm2 -specific siRNA cells under the stimulation of IL-15. 3 d later, cell proliferation and apoptosis were analyzed by Ki67 staining (K) and annexin V staining (L), respectively ( n = 3 per group). Data are shown as mean +- SD and were analyzed by unpaired two-tailed t test (I and J) or one-way ANOVA with Sidak post-test (K and L). Data are representative of at least two independent experiments. ***, P < 0.001. ES,

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Characterization of the cell lines NCCIT and 2102Ep. ( A ) Schematic overview of the hemizygous mutation present in the NCCIT cell line. ( B ) Bar graph displaying the normalized expression (RNA-seq) of MDM2 in the NCCIT and 2102Ep parental and resistant cell lines. ( C ) Western blot showing the protein levels of MDM2 in the NCCIT and 2102Ep parental and resistant cell lines. ( D , E ) Western blot displaying the MDM2 ( D ) and MDM4 ( E ) protein levels after treatment with sublethal cisplatin doses (1 uM) or saline vehicle control. ( F ) Mutational position of TP53 mutations in patients in the MSKCC, J Clin Oncol 2016 data set. The mutation found in NCCIT is highlighted with a blue dot.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 FoxO3a is downregulated in TamR BBCs. ( A ) FoxO3a transcripts were analyzed by real-time PCR in growing MCF-7 and TamR cells. Each sample was normalized vs. its 18S rRNA content and presented as fold enrichment versus MCF-7. The results represent the mean +- s.d. of three independent experiments. *, p < 0.01 vs. untreated. ( B ) Cytoplasmic and nuclear protein extracts from a duplicate set of cells were subjected to WB (30 mug/lane) to evaluate the subcellular localization of FoxO3a. GAPDH and Lamin B (cytosolic and nuclear markers, respectively) were used as loading controls and to assess the quality of the subcellular protein fractionation. ( C ) Immunostaining of FoxO3a expression and localization (green) in MCF-7 and TamR growing cells; nuclear integrity was visualized by DAPI (blue) (400x magnification) ( D , E ) Comparison between AKT ( D ) and MAPK ( E ) signal transduction pathways in MCF-7 and TamR cells. Cells were starved in PRF-SFM for 16 h and then treated or not with EGF (100 nM). Proteins were analyzed by WB, using the indicated antibodies. ( F ) PLA. MCF-7 and TamR cells were seeded in MW8 (Lab-Tek(tm) Chamber Slide System, Nunc(tm)), left to adhere for 48 h, then starved in PRF-SFM and pre-treated with MG132 (20 muM) or left untreated (-). The next day, EGF (100 nM) was added for 30 min where indicated. Antibodies against FoxO3a and MDM2 were used to detect the active complexes. Captures of the stained FoxO3a/MDM2 complexes were analyzed by ImageJ s