MA5-12557

antibody from Invitrogen Antibodies

Targeting: TP53 LFS1, p53

Provider product page for MA5-12557

Western blot

Immunocytochemistry

Flow cytometry

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [182]
Comments [0]
Validations
Western blot [6]
Immunocytochemistry [8]
Flow cytometry [4]
Chromatin Immunoprecipitation [1]
Other assay [15]

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Product number: MA5-12557 - Provider product page
Provider: Invitrogen Antibodies
Product name: p53 Monoclonal Antibody (DO-7)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA5-12557 targets p53 in FACS, ICC/IF, IHC (P), IP, ChIP, and WB applications and shows reactivity with Human and bovine samples. This antibody is not suitable for mouse colon tissue in IHC applications.
Antibody clone number: DO-7
Concentration: 0.05 mg/mL

TP53 protein structure - MA5-12557 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of p53 was performed by loading 20 µg HeLa nuclear cell lysate per lane (untreated or treated with 20 µM Camptothecin for 20 hours) and 7 µl of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). 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. p53 was detected at ~53 kDa using a p53 mouse monoclonal antibody (Product # MA5-12557) at a dilution of 1:1000 in blocking buffer overnight at 4°C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:20,000 for at least 30 minutes at room temperature. HDAC1 was detected using a HDAC1 rabbit polyclonal antibody (Product # PA1-860) at a concentration of 1 µg/mL. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-p53 Monoclonal Antibody (DO-7) (Product # MA5-12557) and a ~53 kDa band corresponding to TP53 was observed across cell lines tested . Whole cell extracts (30 µg lysate) of A-431 (Lane 1), T-47D (Lane 2), SK-O-V3 (Lane 3), HL-60 (Lane 4), U-937 (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:1000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:20000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-p53 Monoclonal Antibody (DO-7) (Product # MA5-12557) and a 53 kDa band corresponding to Cellular tumor antigen p53 was observed across cell lines except HCT 116, SK-OV-3 and HL-60 which are reported to be negative, also it was observed to be induced upon Camptothecin and Doxorubicin treatment in HeLa and MCF7 respectively. Modified whole cell extracts (1%SDS) (30 µg lysate) of HeLa (Lane 1), HeLa treated with Camptothecin (5uM for 20 hours) (Lane 2), MCF7 (Lane 3), MCF7 treated with Doxorubicin (0.5uM for 24 hours) (Lane 4), T-47D (Lane 5), HT-29 (Lane 6), MDA-MB-231 (Lane 7), HCT 116 (Lane 8), SK-O-V3 (Lane 9) and HL-60 (Lane 10) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (1:1000 dilution) 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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of p53 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR718517_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of p53 was performed by loading 30 µg of HeLa Wild Type (Lane 1), Treated HeLa Wild type (Lane 2), HeLa Cas9 (Lane 3), Treated HeLa Cas9 (Lane 4), HeLa Cas9 cells transduced with p53 Lentiviral sgRNA (Lane 5), Treated HeLa Cas9 cells transduced with p53 Lentiviral sgRNA (Lane 6), HeLa p53 KO (Lane 7) and Treated HeLa p53 KO (Lane 8) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 Anti-p53 Monoclonal Antibody (DO-7) (Product # MA5-12557, 1:1,000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to p53. *Treatment done with 5 µM Camptothecin for 20 hrs.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of p53 was performed by loading 20 µg of indicated whole cell lysates per lane and 7 µl of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). 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. p53 was detected at ~53 kDa using a p53 mouse monoclonal antibody (Product # MA5-12557) at a dilution of 1:1000 in blocking buffer overnight at 4°C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:20,000 for at least 30 minutes at room temperature. GAPDH was detected using a GAPDH rabbit polyclonal antibody, HRP conjugate (Product # PA1-987-HRP) at a dilution of 1:1000 overnight at 4°C on a rocking platform.Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of p53 was performed by loading whole-cell lysates of five human osteosarcoma cell lines and non-transformed human osteoblasts (HFOB). p53 was detected at approximately 53 kDa using a p53 monoclonal antibody (Product # MA5-12557) at a dilution of 1:1000 in Blocking Buffer (Product # 37543) overnight at 4°C on a rocking platform, followed by a 1-hour room-temperature incubation of a goat anti-mouse IgG antibody at a dilution of 1:5000. Data courtesy of Thermo Scientific KOL Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Activation of ATM and p53: ATM kinase is auto-phosphorylated in response to DNA damage and is involved in DNA damage induced activation of p53 and cell cycle arrest. This is a multiplex kit, enabling the user to simultaneously measure both phospho-ATM and p53. A549 cells were treated with vehicle (DMSO) or 50 µM etoposide for 24 hrs. After staining cells with Cellomics phospho-ATM/p53 duplex activation kit, images were acquired and analyzed. Z-factor for phospho-ATM = 0.46 ± 0.11 and %COV = 11 ± 3. Z-factor for p53 = 0.44 ± 0.03 and %COV = 13 ± 1. EC50 of etoposide treatment for ATM activation is 19 µM and EC50 for p53 activation is 36 µM. Etoposide is a topoisomerase II inhibitor resulting in DNA strand break.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of p53 (green) in HeLa cells either left untreated (left panel) or treated with 5uM Camptothecin (right panel) for 20 hours. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a p53 monoclonal antibody (Product # MA5-12557) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight Instrument at 20X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of p53 (green) showing staining in the nucleus of A431 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a p53 monoclonal antibody (Product # MA5-12557) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a flourescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

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Experimental details: Immunofluorescent analysis of p53 (green) showing staining in the nucleus of HT29 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a p53 monoclonal antibody (Product # MA5-12557) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a flourescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of TP53 was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with p53 Monoclonal Antibody (DO-7) (Product # MA5-12557) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification.

Supportive validation

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Experimental details: Immunofluorescence analysis of TP53 was performed using 70% confluent log phase T-47D cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with p53 Monoclonal Antibody (DO-7) (Product # MA5-12557) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification.

Supportive validation

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Experimental details: Immunofluorescence analysis of Cellular tumor antigen p53 was performed using 70% confluent log phase MDA-MB-231 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with p53 Monoclonal Antibody (DO-7) (Product # MA5-12557) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear localization. Panel e represents SK-OV-3 cells having no expression of p53. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of p53 (green) in untreated and 5 µM 20 hours Camptothecin treated HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes at -20c, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a p53 mouse monoclonal antibody (Product # MA5-12557) at a dilution of 1:200 in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). F-actin (red) was stained by Dylight 554 Phalloidin (Product #21834) and nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

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Experimental details: Flow cytometry analysis of p53 in A431 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with p53 monoclonal antibody (Product # MA5-12557) at a dilution of 0.5 ug/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

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Experimental details: Flow cytometry analysis of p53 in BAEC cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with p53 monoclonal antibody (Product # MA5-12557) at a dilution of 0.5 ug/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

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Experimental details: Flow cytometry analysis of p53 in CEM cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with p53 monoclonal antibody (Product # MA5-12557) at a dilution of 1 ug/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

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Experimental details: Flow cytometry analysis of p53 was done on HeLa cells. The cells were fixed, permeabilized and stained with a p53 mouse monoclonal antibody (Product # MA5-12557, red histogram) or Mouse IgG2b isotype control (Product # MA5-14447, blue histogram) at a concentration of 10 µg/mL. Black histogram represents negative control unstained cell population. After incubation of the primary antibody on ice for an hour, the cells were stained with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 680 conjugate (Product # A32734) at a dilution of 1:50 for at least 30 minutes on ice. A representative 10,000 cells were acquired for each sample.

Supportive validation

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Experimental details: Fig. 3 DNMT2 silencing-mediated effects on mRNA (A, B) and protein levels (C, D) of cell cycle regulators. HP treatment is also considered. (A, B) The expression of selected genes involved in the regulation of cell cycle. Cells were treated with 100 muM HP for 2 h and then cultured for 24 h. (A) A heat map generated from qRT-PCR data is shown. Hierarchical clustering was created using Genesis 1.7.7 software. (B) HP-mediated up- (red) and downregulation (blue) of cell cycle genes. DeltaDeltaCt values are shown. (C, D) Western blot analysis of the levels of p16, p21 and p53. Data were normalized to beta-actin. Bars indicate SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CTRL (ANOVA and Dunnett's a posteriori test). Proliferatively active fibroblasts were used, namely WI-38 cells at PDLs from 34 to 43 and BJ cells at PDLs from 27 to 43. CTRL, non-transfected control fibroblasts; siFITC, fibroblasts transfected with control FITC-conjugated siRNA; siDNMT2, fibroblasts transfected with DNMT2 siRNA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 3

Supportive validation

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Experimental details: Fig 3 ZIKV NS2A binds to p53. (A, B) U2OS cells were transfected with ZIKV NS2A-Flag. At 18h p.t., the cells were left untreated (A) or stimulated with 10 muM etoposide for 1.5 hrs (B), fixed with methanol and stained with indicated Abs. The images were taken as Z-stack sections and subjected to a digital deconvolution. The scale bar is 50 mum. Arrows point at the cell expressing ZIKV NS2A. (C) Endogenous p53 pull-down. U2OS cells were transfected with pDEST47-ZIKV NS2A-Flag expressing ZIKV NS2A tagged with the C-terminal 3xFlag epitope or left untransfected (UN). At 18 hrs p.t., the cells were stimulated with 10 muM etoposide for 6 hrs. NS2A-Flag was immunoprecipitated with mouse alpha-Flag Ab. Lysates and imunoprecipitates was tested with mouse mab alpha-p53 DO7 and mouse alpha-Flag Abs. (D) ZIKV NS2A pull-down using p53-binding proteins. U2OS cells were transfected with pDEST47-ZIKV NS2A-Flag or left untransfected (UN). At 18 hrs p.t., the cells were stimulated with 10 muM etoposide for 6h. P53 was immunopreciptated with proteins binding to p53 N- or C-terminus. Presence of ZIKV NS2A-Flag and p53 in the lysates and imunoprecipitates was tested with mouse anti-Flag and mouse alpha-p53 DO7 Abs.

Supportive validation

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Experimental details: Fig 6 KSHV orf10 interacts with USP24 and p53. (A, B) U2OS cells were transfected with pDEST47-KSHV orf10-Flag. At 18h p.t., the cells were stimulated with 10muM etoposide for 1.5h (A) or left untreated (B), fixed with methanol, and stained with indicated Abs. Each image represents an individual optical section. The scale bar is 50muM. Arrows point at the cells expressing KSHV orf10. (C) KSHV orf10 colocalizes with USP24. U2OS cells, transfected with pDEST47-KSHV orf10-Flag for 18h were fixed with methanol and stained with indicated alpha-Flag and alpha-USP24 Abs. Each image represents an individual optical section. The scale bar is 50muM. Arrow points at the cell expressing KSHV orf10. (D) USP24 coimmunoprecipitates with KSHV orf10. U2OS cells were transfected with pDEST47-KSHV orf10-Flag expressing orf10 tagged with C-terminal 3xFlag epitope or left untransfected (UN) for 18h. orf10-Flag was immunoprecipitated from the whole cell lysates with mouse alpha-Flag Ab. Presence of USP24 and orf10-Flag in the lysates and coimunoprecipitated fractions was tested with rabbit alpha-USP24 and mouse alpha-Flag Abs. (E) KSHV orf10 coimmunoprecipitates with p53. U2OS cells were transfected with pDEST47-KSHV orf10-Flag or left untransfected (UN) for 18h. P53 was immunopreciptated from nuclear fraction of cell lysates with proteins binding to p53 N- or C-terminus. Presence of orf10-Flag and p53 in the lysates and coimunoprecipitated fractions was tested with mouse alpha-Flag and mouse alph

Supportive validation

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Experimental details: Fig 7 KSHV orf10 interacts with p53 in KSHV infected cells. (A) A293T.219 cells were transfected with pDEST47-KSHV orf10-Flag. At 16h p.t., the cells were induced with 1mM NaB and 25 ng/mL 12-O-tetradecanoylphorbol-13-acetate ( TPA ) for 48h. Stimulation of A293T.219 cells was monitored by RFP expression. Transfected stimulated and unstimulated cells were imaged with bright field and fluorescence microscopy to visualize upregulation of expression of RFP and cytopathic effect (CPE) induced by KSHV replication. Panel A--C show mock-transfected, uninduced cells, panels D--I depict KSHV ORF10-FLAG transfected cells that were either left untreated or induced. Panels (J) KSHV orf10 coimmunoprecipitates with p53. p53 was immunopreciptated from whole cell lysates with antibody binding to p53 C-terminus. Presence of orf10-Flag and p53 in the lysates and co-imunoprecipitated fractions was tested with mouse anti-Flag and mouse anti-p53 DO7 Ab.

Supportive validation

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Experimental details: Figure 4 DNA damage and p53 stabilization after treatment with platinum(II) compounds. A , DNA damage, measured by gammaH2AX immunofluorescence ( green ) after 5 h of treatment with cisplatin and oxaliplatin. B , representative single cell images of A549 cells after treatment with cisplatin and oxaliplatin. C , percentage of gammaH2AX positive nuclei, where a positive threshold was determined by the 90th percentile intensity of the untreated control for each testing day. Imaging was conducted on three separate days, with the exception of ActD, which was only tested on 2 days. Each point represents the percent positive nuclei for a single day and testing condition. D , western blot to detect p53 and loading control beta-actin after Pt(II) treatment at 3 h ( left ) and 24 h ( right ). All scale bars are 10 mum. *** p < 0.001, ns = p > 0.1.

Supportive validation

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Experimental details: Figure 3 Tissue array of mantle cell lymphoma. Immunohistochemistry tissue arrays of MCL patients' samples for ( a ) p53, ( b ) Sox11, and ( c ) Pax5. Nine representative out of 64 of each protein stainings were presented. The red arrow represented p53 positive cells in the MCL cells and zoomed images were above; ( d ) ROC curve analysis of p53 immunohistochemistry as a parameter to discriminate between TP53 mutated and non-mutated samples.

Supportive validation

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Experimental details: Figure 8. Overexpression of miR-532-5p regulates the expression of p53 in HG-induced cells by downregulating CCND1. p53 expression was determined using (A) reverse transcription-quantitative PCR and (B) western blotting. *P

Supportive validation

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Experimental details: Figure 3 Duolink II PLA assay results for HTR8/SVneo cells cultured in normoxic and hypoxic conditions in medium containing 1% serum from preeclamptic ( b , d ) or normotensive ( a , c ) women. Each p53/RSK1 complex in HTR8/SVneo cells is visualised as an individual fluorescence red spot, nuclei were labelled with DAPI. Red spot - complex of p53/RSK1; blue spot - nucleus of cell. Magnification 40x; scale bar 50 um.

Supportive validation

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Experimental details: Figure 6 Western blot results. ( a ) inhibition of p53 by its targeted siRNA; Western blot results for the cytoplasmic fraction, ( b ) the differences in relative level of pNFkB Ser536 to Histone H3 in nucleus of HTR8/SVneo cells. Cells were cultured for 96 h in hypoxic or normoxic conditions with medium containing 1% serum from preeclamptic or normotensive women. For the first 48 h, the cells were incubated only with medium containing the maternal serum. After 48 h and 72 h, the cell medium was supplemented with the following factors: none i.e., cells without siRNA (1), non-targeting siRNA (2) and targeted siRNA against mRNA coding for p53 (3). The relative level of pNFkB Ser536 to Histone H3 in the nucleus was analyzed. Values are presented as mean +- SEM; ( n = 3experiments). The difference in pNFkB Ser536 level between cells stimulated by negative and targeted siRNA was calculated by the Student's t -test; ## p < 0.01. The difference in pNFkB Ser536 level between the four culture types (i.e., oxygen tension and serum type) which were not stimulated with siRNA were analyzed by ANOVA with the Bonferroni post hoc test; ** p < 0.01, *** p < 0.001. ( c ) Western blot results for nuclear fraction containing pNFkB Ser536 and Histone H3, as a loading control.

Supportive validation

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Experimental details: Figure 5. Garcinol regulates the expression of cell cycle-related proteins in EC cells. Effect of garcinol on the expression of cell cycle-regulated genes in EC cells were detected by western blotting. ISH and HEC-1B cells were cultured for 24 h and then treated with and without garcinol (0, 1, 5, 10 and 20 uM) for another 24 h. (A) Total protein was collected, and western blot analyses were performed for p53, p21, cyclin D1, cyclin B1, CDK2, CDK4. beta-actin was used as alpha loading control. (B) Band density was analyzed by ImageJ, and the results are expressed as the mean +- SD, n=3. *P

Supportive validation

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Experimental details: Fig. 3 Cell Cycle and TP53 gene signatures, TP53 mutational analysis, and immunohistochemical validation. Detailed examination of the proliferation, cell death, and DNA damage pathways, as identified by RNA expression profiling described in Fig. 2 , and IHC examination of these pathways. Depiction of a two additional GSEA enrichment plots for cell cycle. b Single sample cell cycle score determined from RNA expression values from the indicated genes (PPBC n = 9, NPBC n = 7). Data are presented as a minimum to maximum with median value marked by a line within the depicted interquartile range, and p value determined by Students' unpaired two-tailed t test with Welch correction. c Examples of immunohistochemical (IHC) evaluation of KI67-positive (brown color) protein expression (left), with quantification of KI67 signal evaluated as the proportion of nuclei (right, PPBC n = 15, NPBC n = 15) Data are presented as mean values +-SEM, and p value determined by Students' unpaired two-tailed t test with Welch correction. Samples evaluated by RNA Seq are depicted by circles and pseq refers to p values for these samples only, while expanded cases for IHC are depicted by squares and p values reflect values for the whole cohort. d GSEA analysis assessments of cell death (left) or DNA damage and repair associated gene sets (TP53, right) (PPBC n = 9, NPBC n = 7). e Flow diagram outlining computational steps and results for prediction of the presence of wildtype (WT) or mutant (MUT) TP53 gene

Supportive validation

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Experimental details: Figure 4 (A) Transcription factor profile of DEGs with photodynamic intervention (dose 20 J/cm 2 ) using TRRUST database. (B) DEGs overlap among E2F1, SP1, and TP53 transcription factors. (C) Immunostaining of p53 nuclear translation after photodynamic intervention (20 J/cm 2 ). Scale bar is 10 mum.

Supportive validation

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Experimental details: Figure 4 TCGA classification by immunohistochemistry and EBER for the three NHPH positive gastric cancer. ( A , G , M ) Hematoxylin and eosin staining for gastric cancer in Patients 1, 2, and 3, respectively. ( B , H , O ) MLH1 staining was positive for all three gastric cancers and no microsatellite instability was indicated. ( C , I , P ) p53 staining was positive and p53 was aberrantly expressed in all three gastric cancers. ( D , J , Q ) p53 staining in the non-cancerous part of gastric mucosa in each patient. P53 was not aberrantly expressed in normal regions. ( E , K , R ) E-cadherin was positive in all three cancers. ( F , L , S ) EBER in situ hybridization. Epstain-Barr virus was negative in all three stomachs. Inset in ( F ) shows positive control. ( N ) shows the surrounding non-cancerous parts. Taken together, all three gastric cancers were classified into the Chromosomal Instability (CIN) Type.

Supportive validation

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Experimental details: 10.1371/journal.pone.0267615.g002 Fig 2 p53 involvement in the ubiquitylation of RNAPII following ActD treatment is a ubiquitous process. ( A ) Tandem ubiquitin-binding entities (TUBEs) pull-down followed by Western blot detection was performed in HCT116 p53+/+ (left panel) and p53-/- (right panel) cells. TUBEs experiment was accomplished under physiological conditions as well as 8 h and 24 h following ActD treatments. From the pulled-down ubiquitylated protein pool, polyubiquitylated S2P RNAPII (ub-S2P RNAPII) was detected with anti-S2P RNAPII antibody. *: monoubiquitylated S2P RNAPII, **: polyubiquitylated S2P RNAPII ( B ) Western blot experiment on whole cell lysates of HCT116 p53+/+ (left panel) and p53-/- (right panel), which were used for TUBEs pull-down assay. Total protein level changes of S2P RNAPII and p53 following 8 h and 24 h ActD treatments were immunodetected using specific antibodies. Ponceau staining was applied to detect the equal loading of the input samples. ( C ) Tandem ubiquitin-binding entities (TUBEs) pull-down followed by Western blot detection was performed in non-targeting and p53 silencing siRNA-transfected (referred to as siSCR and sip53, respectively) U2OS cells. In U2OS cells, TUBEs assay was performed under physiological conditions, 6 h and 24 h following ActD treatments. From the pulled-down ubiquitylated protein pool, polyubiquitylated S2P RNAPII (ub-S2P RNAPII) was detected with anti-S2P RNAPII antibody. ( D ) Western blot experiment on whol

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 MNPQ-mediated changes in the levels (A) and localization (B) of cell cycle inhibitors, namely p21 and p53. (A) Western blot analysis of the levels of p21 and p53. Data were normalized to beta-actin. (B) Immunofluorescence analysis of cellular localization of p21 and p53. Representative histograms are presented (left). Samples without HP treatment are red and samples with HP treatment are green. Nuclear signals of p21 and p53 are denoted with horizontal lines (Nuc.). Representative images are also shown (right). Ch01, bright field (BF); Ch05, nucleus staining using propidium iodide staining (red, PI); Ch02, immunostaining of p21 and p53 using secondary antibodies conjugated to Alexa Fluor Plus 488 (green, AFP 488); Ch05/Ch02, a merged image of Ch05 and Ch02. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to CTRL, ### p < 0.001, ## p < 0.01, # p < 0.05 compared to HP-treated control (ANOVA and Dunnett's a posteriori test). CTRL , control conditions; Q , quercetin; MNP , magnetite nanoparticles; MNPQ , quercetin surface functionalized magnetite nanoparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 4