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p53 dominant negative r175h mutant pcw107 v5 (Addgene inc)

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Addgene inc p53 dominant negative r175h mutant pcw107 v5

SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), <t>P53</t> (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

P53 Dominant Negative R175h Mutant Pcw107 V5, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy"

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-025-02640-y

Figure Legend Snippet: SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

Techniques Used: Activity Assay, Stable Transfection, Expressing, Control, Staining

P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

Figure Legend Snippet: P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

Techniques Used: Phospho-proteomics, Stable Transfection, Expressing, Control, Staining

SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

Figure Legend Snippet: SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

Techniques Used: Activity Assay

Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

Figure Legend Snippet: Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

Techniques Used: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation, Staining, Flow Cytometry

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Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

Techniques: Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation, Staining, Flow Cytometry

Galectin 7 binding to PD-1 leads to SHP-2 recruitment and suppression of NFAT. ( a ) Jurkat cells transduced with lentivirus encoding Fc tagged human PD1 were treated with BSA or Galectin-7 (1 μM), and the interaction of PD1 and shp2 was analyzed with co-IP. ( b ) Jurkat NFAT reporter cells were transfected with control siRNA or siRNA against PD1. Another set of Jurkat NFAT reporter cells was transduced to overexpress WT or dominant negative shp2 mutant (SHP2CS). Cells were cultured in a plate coated with anti-CD3 in the presence of BSA or Galectin-7 (1 μM). The activation of NFAT signaling was analyzed by quantifying the luciferase activity using a plate reader. (t-test, ** p = 0.0023, *** p = 0.00078), (n = 3). Error bars show mean ± SD.

Journal: Scientific Reports

Article Title: Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1

doi: 10.1038/s41598-024-57162-3

Figure Lengend Snippet: Galectin 7 binding to PD-1 leads to SHP-2 recruitment and suppression of NFAT. ( a ) Jurkat cells transduced with lentivirus encoding Fc tagged human PD1 were treated with BSA or Galectin-7 (1 μM), and the interaction of PD1 and shp2 was analyzed with co-IP. ( b ) Jurkat NFAT reporter cells were transfected with control siRNA or siRNA against PD1. Another set of Jurkat NFAT reporter cells was transduced to overexpress WT or dominant negative shp2 mutant (SHP2CS). Cells were cultured in a plate coated with anti-CD3 in the presence of BSA or Galectin-7 (1 μM). The activation of NFAT signaling was analyzed by quantifying the luciferase activity using a plate reader. (t-test, ** p = 0.0023, *** p = 0.00078), (n = 3). Error bars show mean ± SD.

Article Snippet: Galectin 7 suppressed the NFAT transcription activity, and the inhibition of PD-1 using siRNA or blockade of SHP-2 activity using a dominant-negative SHP-2 mutant (SHP2CS) blunted the effect of galectin 7 (Fig. b).

Techniques: Binding Assay, Transduction, Co-Immunoprecipitation Assay, Transfection, Control, Dominant Negative Mutation, Mutagenesis, Cell Culture, Activation Assay, Luciferase, Activity Assay

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