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Cell Signaling Technology Inc polyclonal antibodies against mdm2

PIP 2 associates with <t>MDM2</t> in the nucleus in response to stress. A , MDM2 recombinant protein (0.1 μg) was incubated with 1.0 nM liposomes containing the indicated phosphoinositides. Liposomes were sedimented, and the associated MDM2 was analyzed by immunoblotting (IB). The MDM2 IB intensity was quantified, and the graph is shown as mean ± standard deviation(s.d.) of n = 4 independent experiments. B , confocal images of immunofluorescence (IF) staining against PIP 2 and MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin (Cis) for 24 h. MDM2 and PIP 2 antibodies were used for the immunostaining study: scale bars, 5 μm. C , MDA-MB-231, and MDA-MB-468 cells were treated with 30 μM cisplatin or vehicle for 24 h and then processed for immunoprecipitation (IP) of MDM2 and fluorescence IB. Fluorescence IP−IB detects stress-induced PIP 2 association with endogenous MDM2. The PIP 2 IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. D , proximity ligation assay (PLA) of MDM2-PIP 2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of MDM2-PIP 2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. E , HEK293FT cells transiently transfected with MDM2 were treated with vehicle or 25 μCi/ml 3 H-myo-inositol to metabolic labeling. Cells were treated with vehicle or 30 μM cisplatin for 24 h, then processed for Coomassie blue staining and IP of MDM2 and fluorescence IB. The gel was sliced by molecular weight corresponding to the adjacent graph and analyzed by liquid scintillation counting (LSC). n = 3 slices pooled from 3 independent experiments, 6 slices per experiment.

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1) Product Images from "Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex"

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2025.110527

PIP 2 associates with MDM2 in the nucleus in response to stress. A , MDM2 recombinant protein (0.1 μg) was incubated with 1.0 nM liposomes containing the indicated phosphoinositides. Liposomes were sedimented, and the associated MDM2 was analyzed by immunoblotting (IB). The MDM2 IB intensity was quantified, and the graph is shown as mean ± standard deviation(s.d.) of n = 4 independent experiments. B , confocal images of immunofluorescence (IF) staining against PIP 2 and MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin (Cis) for 24 h. MDM2 and PIP 2 antibodies were used for the immunostaining study: scale bars, 5 μm. C , MDA-MB-231, and MDA-MB-468 cells were treated with 30 μM cisplatin or vehicle for 24 h and then processed for immunoprecipitation (IP) of MDM2 and fluorescence IB. Fluorescence IP−IB detects stress-induced PIP 2 association with endogenous MDM2. The PIP 2 IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. D , proximity ligation assay (PLA) of MDM2-PIP 2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of MDM2-PIP 2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. E , HEK293FT cells transiently transfected with MDM2 were treated with vehicle or 25 μCi/ml 3 H-myo-inositol to metabolic labeling. Cells were treated with vehicle or 30 μM cisplatin for 24 h, then processed for Coomassie blue staining and IP of MDM2 and fluorescence IB. The gel was sliced by molecular weight corresponding to the adjacent graph and analyzed by liquid scintillation counting (LSC). n = 3 slices pooled from 3 independent experiments, 6 slices per experiment.

Figure Legend Snippet: PIP 2 associates with MDM2 in the nucleus in response to stress. A , MDM2 recombinant protein (0.1 μg) was incubated with 1.0 nM liposomes containing the indicated phosphoinositides. Liposomes were sedimented, and the associated MDM2 was analyzed by immunoblotting (IB). The MDM2 IB intensity was quantified, and the graph is shown as mean ± standard deviation(s.d.) of n = 4 independent experiments. B , confocal images of immunofluorescence (IF) staining against PIP 2 and MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin (Cis) for 24 h. MDM2 and PIP 2 antibodies were used for the immunostaining study: scale bars, 5 μm. C , MDA-MB-231, and MDA-MB-468 cells were treated with 30 μM cisplatin or vehicle for 24 h and then processed for immunoprecipitation (IP) of MDM2 and fluorescence IB. Fluorescence IP−IB detects stress-induced PIP 2 association with endogenous MDM2. The PIP 2 IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. D , proximity ligation assay (PLA) of MDM2-PIP 2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of MDM2-PIP 2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. E , HEK293FT cells transiently transfected with MDM2 were treated with vehicle or 25 μCi/ml 3 H-myo-inositol to metabolic labeling. Cells were treated with vehicle or 30 μM cisplatin for 24 h, then processed for Coomassie blue staining and IP of MDM2 and fluorescence IB. The gel was sliced by molecular weight corresponding to the adjacent graph and analyzed by liquid scintillation counting (LSC). n = 3 slices pooled from 3 independent experiments, 6 slices per experiment.

Techniques Used: Recombinant, Incubation, Liposomes, Western Blot, Standard Deviation, Immunofluorescence, Staining, Immunostaining, Immunoprecipitation, Fluorescence, Proximity Ligation Assay, Transfection, Labeling, Molecular Weight

PIPKIα interacts with MDM2 and regulates PIP 2 association. A and B , Co-IP of MDM2 with PIPKIα from MDA-MB-231 ( A ) and MDA-MB-468 cells ( B ) treated with 30 μM cisplatin or vehicle for 24 h. Data shown represent three independent experiments. The PIPKIα IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Veh, vehicle; Cis, cisplatin-treated; short, short-time exposure; long, long-time exposure. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The MDM2, PIPKIα, PIPKIγ, PIPKIIα, PIPKIIβ, p53, and GAPDH IPed by MDM2 were analyzed by IB. short, short-time exposure; long, long-time exposure. D , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2 or 4 μg of PIPKIα protein. MDM2 was pulled down, and the binding with PIPKIα was analyzed with an anti-PIPKIα antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. E , Quantification of nuclear PLA of PIPKIα-MDM2 in MDA-MB-231 cells treated with vehicle, cisplatin, or cisplatin plus siRNAs targeting PIPKIα for KD. For cisplatin treatment, 30 μM cisplatin was added for 24 h. PIPKIα KD was achieved using siRNAs for 24 h, followed by 24 h cisplatin treatment. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. Veh, vehicle; Cis, cisplatin-treated, KD; PIPKIα KD. F , MDA-MB-231 cells were transfected with siRNAs targeting PIPKIα. After 24 h of transfection, cells were treated with 30 μM cisplatin for 24 h. IB analyzed the expression of the indicated proteins, IBs were quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Cis, cisplatin-treated; KD, knockdown; Mock, empty vector.

Figure Legend Snippet: PIPKIα interacts with MDM2 and regulates PIP 2 association. A and B , Co-IP of MDM2 with PIPKIα from MDA-MB-231 ( A ) and MDA-MB-468 cells ( B ) treated with 30 μM cisplatin or vehicle for 24 h. Data shown represent three independent experiments. The PIPKIα IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Veh, vehicle; Cis, cisplatin-treated; short, short-time exposure; long, long-time exposure. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The MDM2, PIPKIα, PIPKIγ, PIPKIIα, PIPKIIβ, p53, and GAPDH IPed by MDM2 were analyzed by IB. short, short-time exposure; long, long-time exposure. D , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2 or 4 μg of PIPKIα protein. MDM2 was pulled down, and the binding with PIPKIα was analyzed with an anti-PIPKIα antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. E , Quantification of nuclear PLA of PIPKIα-MDM2 in MDA-MB-231 cells treated with vehicle, cisplatin, or cisplatin plus siRNAs targeting PIPKIα for KD. For cisplatin treatment, 30 μM cisplatin was added for 24 h. PIPKIα KD was achieved using siRNAs for 24 h, followed by 24 h cisplatin treatment. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. Veh, vehicle; Cis, cisplatin-treated, KD; PIPKIα KD. F , MDA-MB-231 cells were transfected with siRNAs targeting PIPKIα. After 24 h of transfection, cells were treated with 30 μM cisplatin for 24 h. IB analyzed the expression of the indicated proteins, IBs were quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Cis, cisplatin-treated; KD, knockdown; Mock, empty vector.

Techniques Used: Co-Immunoprecipitation Assay, Recombinant, Incubation, Binding Assay, Transfection, Expressing, Knockdown, Plasmid Preparation

MDM2 associates with sHSPs in the nucleus. A and B , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2, or 4 μg of αBC ( A ) and HSP27 ( B ) proteins. MDM2 was pulled down, and the association with αBC and HSP27 was analyzed using anti-αBC and anti-HSP27 antibodies. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. IB analyzed the MDM2, αBC, HSP27, and GAPDH IPed by MDM2. Veh, vehicle; Cis, cisplatin-treated. D and E , Confocal images of IF staining against αBC ( D ) and HSP27 ( E ) along with MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear levels of MDM2, αBC, and HSP27 were normalized to vehicle-treated cells, and their nuclear co-localization was determined by immunofluorescence. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. F and G , PLA of αBC–MDM2 ( F ) and HSP27-MDM2 ( G ) in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of αBC–MDM2 and HSP27-MDM2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment.

Figure Legend Snippet: MDM2 associates with sHSPs in the nucleus. A and B , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2, or 4 μg of αBC ( A ) and HSP27 ( B ) proteins. MDM2 was pulled down, and the association with αBC and HSP27 was analyzed using anti-αBC and anti-HSP27 antibodies. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. IB analyzed the MDM2, αBC, HSP27, and GAPDH IPed by MDM2. Veh, vehicle; Cis, cisplatin-treated. D and E , Confocal images of IF staining against αBC ( D ) and HSP27 ( E ) along with MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear levels of MDM2, αBC, and HSP27 were normalized to vehicle-treated cells, and their nuclear co-localization was determined by immunofluorescence. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. F and G , PLA of αBC–MDM2 ( F ) and HSP27-MDM2 ( G ) in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of αBC–MDM2 and HSP27-MDM2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment.

Techniques Used: Recombinant, Incubation, Co-Immunoprecipitation Assay, Staining, Immunofluorescence

PIP 2 regulates the interaction of MDM2, sHSPs and p53 and controls MDM2 stability. A and B , 0.1 μg of MDM2 recombinant protein and 0.1 μg of αBC ( A ) or HSP27 ( B ) protein were incubated with 0, 0.5, 1, 2, or 5 μM PIP 2 . MDM2 was pulled down, and the associated αBC or HSP27 was analyzed with an anti-αBC or an anti-HSP27 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-468 cells were transfected with siRNAs for αBC or HSP27 for 48 h. An empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB MDM2 IBs were quantified, and the graph is shown as mean ± s.d. of n = 4 independent experiments. KD, knockdown. D , MDA-MB-468 cells were transfected with siRNAs for αBC for 48 h. Before harvesting, cells were treated with 10 μM of MG132 for 4 h. Empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB, and MDM2 IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. E , recombinant MDM2 protein (0.1 μg) and GST-tagged p53 WT or PIP2-binding-defective p53 6Q mutant (p53 6Q, 0.1 μg) were incubated with 0, 1, 5, 10, or 50 μM PIP 2 . MDM2 was pulled down, and the associated p53 was analyzed with an anti-p53 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. p53 WT, wild type p53; p53 6Q, p53 6Q mutant.

Figure Legend Snippet: PIP 2 regulates the interaction of MDM2, sHSPs and p53 and controls MDM2 stability. A and B , 0.1 μg of MDM2 recombinant protein and 0.1 μg of αBC ( A ) or HSP27 ( B ) protein were incubated with 0, 0.5, 1, 2, or 5 μM PIP 2 . MDM2 was pulled down, and the associated αBC or HSP27 was analyzed with an anti-αBC or an anti-HSP27 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-468 cells were transfected with siRNAs for αBC or HSP27 for 48 h. An empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB MDM2 IBs were quantified, and the graph is shown as mean ± s.d. of n = 4 independent experiments. KD, knockdown. D , MDA-MB-468 cells were transfected with siRNAs for αBC for 48 h. Before harvesting, cells were treated with 10 μM of MG132 for 4 h. Empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB, and MDM2 IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. E , recombinant MDM2 protein (0.1 μg) and GST-tagged p53 WT or PIP2-binding-defective p53 6Q mutant (p53 6Q, 0.1 μg) were incubated with 0, 1, 5, 10, or 50 μM PIP 2 . MDM2 was pulled down, and the associated p53 was analyzed with an anti-p53 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. p53 WT, wild type p53; p53 6Q, p53 6Q mutant.

Techniques Used: Recombinant, Incubation, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Binding Assay, Mutagenesis

PIP 2 and sHSPs regulate the MDM2-p53 interaction. A and B , 0.1 μg of MDM2 and GST-p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC ( A ) or HSP27 ( B ) in the presence or absence of 10 μM of PIP 2 . After pulling down with MDM2 antibody-conjugated beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. sup, supernatant; pel, pellet. C , 0.5 μg Bacteria (His-tagged) and mammalian-expressed (FLAG-tagged) MDM2 ( A ) and p53 ( B ) proteins were analyzed by fluorescence IB. Fluorescence IB detects purified proteins and PIP 2 stably associated to proteins. The MDM2 and PIP 2 association is shown in the merged image. D , recombinant MDM2 protein (0.1 μg) and FLAG-, His-p53, or GST-p53 6Q mutant proteins were incubated with 0, 1, 5, or 10 μM PIP 2 . MDM2 was pulled down, and the associated different p53 was analyzed with an anti-p53 antibody. The graphs are shown bound p53 normalized to the strongest signal (His-p53 with 10 μM PIP 2 ) as mean ± s.d. of n = 3 independent experiments. p53 6Q, p53 6Q mutant. E , 0.1 μg of FLAG-MDM2 and p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC or HSP27. After pulling down with MDM2 beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. FLAG-tagged proteins; mammalian-derived proteins.

Figure Legend Snippet: PIP 2 and sHSPs regulate the MDM2-p53 interaction. A and B , 0.1 μg of MDM2 and GST-p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC ( A ) or HSP27 ( B ) in the presence or absence of 10 μM of PIP 2 . After pulling down with MDM2 antibody-conjugated beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. sup, supernatant; pel, pellet. C , 0.5 μg Bacteria (His-tagged) and mammalian-expressed (FLAG-tagged) MDM2 ( A ) and p53 ( B ) proteins were analyzed by fluorescence IB. Fluorescence IB detects purified proteins and PIP 2 stably associated to proteins. The MDM2 and PIP 2 association is shown in the merged image. D , recombinant MDM2 protein (0.1 μg) and FLAG-, His-p53, or GST-p53 6Q mutant proteins were incubated with 0, 1, 5, or 10 μM PIP 2 . MDM2 was pulled down, and the associated different p53 was analyzed with an anti-p53 antibody. The graphs are shown bound p53 normalized to the strongest signal (His-p53 with 10 μM PIP 2 ) as mean ± s.d. of n = 3 independent experiments. p53 6Q, p53 6Q mutant. E , 0.1 μg of FLAG-MDM2 and p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC or HSP27. After pulling down with MDM2 beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. FLAG-tagged proteins; mammalian-derived proteins.

Techniques Used: Recombinant, Incubation, Bacteria, Fluorescence, Purification, Stable Transfection, Mutagenesis, Derivative Assay

PIP 2 and sHSPs regulate the ubiquitination function of MDM2. A , for MDM2 autoubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM) for 1 h. Ubiquitin, MDM2, αBC and HSP27 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. B , for in vitro ubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of His- and FLAG-tagged MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with 1 μM of His- and FLAG-tagged p53 for 1 h. For IP, samples were incubated with anti-p53-conjugated agarose overnight. Ubiquitin, MDM2, and p53 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-231 cells were transfected with siRNAs for PIPKIα and treated with vehicle or 30 μM cisplatin for 24 h. Empty vector (Mock) was used as a negative control. After cells were treated with 10 μM of MG132 for 4 h, cells were harvested for IP of MDM2 and p53. Expression of the indicated proteins was analyzed by IB, and ubiquitin IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown; UT, untreated; Cis, cisplatin treated; Cis/MG132, Cisplatin/MG132 treated. D , MDA-MB-468 cells were transfected with siRNAs for αBC and HSP27 for 48 h in absence or presence of MG132. Empty vector (Mock) was used as a negative control. Cells were processed for IP of MDM2. IB was used to analyze indicated proteins, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown. E , a model of PIP 2 regulation of the interaction between MDM2 and sHSPs, which differentially control the stability of MDM2 and function of of MDM2. In the presence of PIPKIα, PIP 2 is linked to MDM2, which recruits αBC to MDM2 to stabilize it and enhances p53 binding. Conversely, in the absence of PIPKIα and PIP 2 , HSP27 is recruited to MDM2, which increases the ubiquitin E3 ligase activity of MDM2.

Figure Legend Snippet: PIP 2 and sHSPs regulate the ubiquitination function of MDM2. A , for MDM2 autoubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM) for 1 h. Ubiquitin, MDM2, αBC and HSP27 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. B , for in vitro ubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of His- and FLAG-tagged MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with 1 μM of His- and FLAG-tagged p53 for 1 h. For IP, samples were incubated with anti-p53-conjugated agarose overnight. Ubiquitin, MDM2, and p53 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-231 cells were transfected with siRNAs for PIPKIα and treated with vehicle or 30 μM cisplatin for 24 h. Empty vector (Mock) was used as a negative control. After cells were treated with 10 μM of MG132 for 4 h, cells were harvested for IP of MDM2 and p53. Expression of the indicated proteins was analyzed by IB, and ubiquitin IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown; UT, untreated; Cis, cisplatin treated; Cis/MG132, Cisplatin/MG132 treated. D , MDA-MB-468 cells were transfected with siRNAs for αBC and HSP27 for 48 h in absence or presence of MG132. Empty vector (Mock) was used as a negative control. Cells were processed for IP of MDM2. IB was used to analyze indicated proteins, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown. E , a model of PIP 2 regulation of the interaction between MDM2 and sHSPs, which differentially control the stability of MDM2 and function of of MDM2. In the presence of PIPKIα, PIP 2 is linked to MDM2, which recruits αBC to MDM2 to stabilize it and enhances p53 binding. Conversely, in the absence of PIPKIα and PIP 2 , HSP27 is recruited to MDM2, which increases the ubiquitin E3 ligase activity of MDM2.

Techniques Used: Ubiquitin Proteomics, Incubation, In Vitro, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Control, Binding Assay, Activity Assay

Bioprocessing:

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex
Article Snippet: Recombinant GST-tagged p53 (#14-865) was purchased from MilliporeSigma. .. Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study. ..

Article Title: Regulation of the MDM2-p53 Nexus by a Nuclear Phosphoinositide and Small Heat Shock Protein Complex.
Article Snippet: Recombinant GST-tagged p53 (#14-865) was purchased from MilliporeSigma. .. Antibodies and reagents Jo urn al Pr e-p roo f 11 Monoclonal antibodies against PIP2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study. ..

Article Title: Regulation of the MDM2-p53 Nexus by a Nuclear Phosphoinositide and Small Heat Shock Protein Complex
Article Snippet: Recombinant GST-tagged p53 (#14-865) was purchased from MilliporeSigma. .. Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), PTEN (clone D4,3, #9188, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), PITPNB (#NBP2-19841, Novus Biologicals), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study. ..

Ubiquitin Proteomics:

Image Search Results

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 associates with MDM2 in the nucleus in response to stress. A , MDM2 recombinant protein (0.1 μg) was incubated with 1.0 nM liposomes containing the indicated phosphoinositides. Liposomes were sedimented, and the associated MDM2 was analyzed by immunoblotting (IB). The MDM2 IB intensity was quantified, and the graph is shown as mean ± standard deviation(s.d.) of n = 4 independent experiments. B , confocal images of immunofluorescence (IF) staining against PIP 2 and MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin (Cis) for 24 h. MDM2 and PIP 2 antibodies were used for the immunostaining study: scale bars, 5 μm. C , MDA-MB-231, and MDA-MB-468 cells were treated with 30 μM cisplatin or vehicle for 24 h and then processed for immunoprecipitation (IP) of MDM2 and fluorescence IB. Fluorescence IP−IB detects stress-induced PIP 2 association with endogenous MDM2. The PIP 2 IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. D , proximity ligation assay (PLA) of MDM2-PIP 2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of MDM2-PIP 2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. E , HEK293FT cells transiently transfected with MDM2 were treated with vehicle or 25 μCi/ml 3 H-myo-inositol to metabolic labeling. Cells were treated with vehicle or 30 μM cisplatin for 24 h, then processed for Coomassie blue staining and IP of MDM2 and fluorescence IB. The gel was sliced by molecular weight corresponding to the adjacent graph and analyzed by liquid scintillation counting (LSC). n = 3 slices pooled from 3 independent experiments, 6 slices per experiment.

Article Snippet: Monoclonal antibodies against PIP 2 (clone 2C11, #Z-P045, Echelon Biosciences), PIP 2 (clone KT10, #MSBS2283, MilliporeSigma), p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), HSP27 (clone F-4, #SC-13132, Santa Cruz Biotechnology), αBC (D6S9E, #45844, Cell Signaling), PIPKIIα (PIP4K2A, clone D83C1, #5527, Cell Signaling), GAPDH (clone 0411, #SC-47724, Santa Cruz Biotechnology), and polyclonal antibodies against MDM2 (clone D1V2Z, #86934, Cell Signaling), MDM2 (#AF1244, R&D Systems), αBC (#ab13497, Abcam), PIPKIα (PIP5K1A, #9693, Cell Signaling), PIPKIγ (PIP5K1C, #3296, Cell Signaling), PIPKIIβ (PIP4K2B, #9694, Cell Signaling), FOXO3a (clone 75D8, #2497, Cell Signaling), Histone H2A (#2578, Cell Signaling), Histone H2B (clone D2H6, #12364, Cell Signaling), Ubiquitin (P37, #58395, Cell Signaling) were utilized in this study.

Techniques: Recombinant, Incubation, Liposomes, Western Blot, Standard Deviation, Immunofluorescence, Staining, Immunostaining, Immunoprecipitation, Fluorescence, Proximity Ligation Assay, Transfection, Labeling, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIPKIα interacts with MDM2 and regulates PIP 2 association. A and B , Co-IP of MDM2 with PIPKIα from MDA-MB-231 ( A ) and MDA-MB-468 cells ( B ) treated with 30 μM cisplatin or vehicle for 24 h. Data shown represent three independent experiments. The PIPKIα IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Veh, vehicle; Cis, cisplatin-treated; short, short-time exposure; long, long-time exposure. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The MDM2, PIPKIα, PIPKIγ, PIPKIIα, PIPKIIβ, p53, and GAPDH IPed by MDM2 were analyzed by IB. short, short-time exposure; long, long-time exposure. D , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2 or 4 μg of PIPKIα protein. MDM2 was pulled down, and the binding with PIPKIα was analyzed with an anti-PIPKIα antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. E , Quantification of nuclear PLA of PIPKIα-MDM2 in MDA-MB-231 cells treated with vehicle, cisplatin, or cisplatin plus siRNAs targeting PIPKIα for KD. For cisplatin treatment, 30 μM cisplatin was added for 24 h. PIPKIα KD was achieved using siRNAs for 24 h, followed by 24 h cisplatin treatment. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. Veh, vehicle; Cis, cisplatin-treated, KD; PIPKIα KD. F , MDA-MB-231 cells were transfected with siRNAs targeting PIPKIα. After 24 h of transfection, cells were treated with 30 μM cisplatin for 24 h. IB analyzed the expression of the indicated proteins, IBs were quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Cis, cisplatin-treated; KD, knockdown; Mock, empty vector.

Techniques: Co-Immunoprecipitation Assay, Recombinant, Incubation, Binding Assay, Transfection, Expressing, Knockdown, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: MDM2 associates with sHSPs in the nucleus. A and B , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2, or 4 μg of αBC ( A ) and HSP27 ( B ) proteins. MDM2 was pulled down, and the association with αBC and HSP27 was analyzed using anti-αBC and anti-HSP27 antibodies. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. IB analyzed the MDM2, αBC, HSP27, and GAPDH IPed by MDM2. Veh, vehicle; Cis, cisplatin-treated. D and E , Confocal images of IF staining against αBC ( D ) and HSP27 ( E ) along with MDM2 in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear levels of MDM2, αBC, and HSP27 were normalized to vehicle-treated cells, and their nuclear co-localization was determined by immunofluorescence. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. F and G , PLA of αBC–MDM2 ( F ) and HSP27-MDM2 ( G ) in MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclear PLA foci of αBC–MDM2 and HSP27-MDM2 were quantified. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment.

Techniques: Recombinant, Incubation, Co-Immunoprecipitation Assay, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 regulates the interaction of MDM2, sHSPs and p53 and controls MDM2 stability. A and B , 0.1 μg of MDM2 recombinant protein and 0.1 μg of αBC ( A ) or HSP27 ( B ) protein were incubated with 0, 0.5, 1, 2, or 5 μM PIP 2 . MDM2 was pulled down, and the associated αBC or HSP27 was analyzed with an anti-αBC or an anti-HSP27 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-468 cells were transfected with siRNAs for αBC or HSP27 for 48 h. An empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB MDM2 IBs were quantified, and the graph is shown as mean ± s.d. of n = 4 independent experiments. KD, knockdown. D , MDA-MB-468 cells were transfected with siRNAs for αBC for 48 h. Before harvesting, cells were treated with 10 μM of MG132 for 4 h. Empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB, and MDM2 IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. E , recombinant MDM2 protein (0.1 μg) and GST-tagged p53 WT or PIP2-binding-defective p53 6Q mutant (p53 6Q, 0.1 μg) were incubated with 0, 1, 5, 10, or 50 μM PIP 2 . MDM2 was pulled down, and the associated p53 was analyzed with an anti-p53 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. p53 WT, wild type p53; p53 6Q, p53 6Q mutant.

Techniques: Recombinant, Incubation, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the MDM2-p53 interaction. A and B , 0.1 μg of MDM2 and GST-p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC ( A ) or HSP27 ( B ) in the presence or absence of 10 μM of PIP 2 . After pulling down with MDM2 antibody-conjugated beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. sup, supernatant; pel, pellet. C , 0.5 μg Bacteria (His-tagged) and mammalian-expressed (FLAG-tagged) MDM2 ( A ) and p53 ( B ) proteins were analyzed by fluorescence IB. Fluorescence IB detects purified proteins and PIP 2 stably associated to proteins. The MDM2 and PIP 2 association is shown in the merged image. D , recombinant MDM2 protein (0.1 μg) and FLAG-, His-p53, or GST-p53 6Q mutant proteins were incubated with 0, 1, 5, or 10 μM PIP 2 . MDM2 was pulled down, and the associated different p53 was analyzed with an anti-p53 antibody. The graphs are shown bound p53 normalized to the strongest signal (His-p53 with 10 μM PIP 2 ) as mean ± s.d. of n = 3 independent experiments. p53 6Q, p53 6Q mutant. E , 0.1 μg of FLAG-MDM2 and p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC or HSP27. After pulling down with MDM2 beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. FLAG-tagged proteins; mammalian-derived proteins.

Techniques: Recombinant, Incubation, Bacteria, Fluorescence, Purification, Stable Transfection, Mutagenesis, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the ubiquitination function of MDM2. A , for MDM2 autoubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM) for 1 h. Ubiquitin, MDM2, αBC and HSP27 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. B , for in vitro ubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of His- and FLAG-tagged MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with 1 μM of His- and FLAG-tagged p53 for 1 h. For IP, samples were incubated with anti-p53-conjugated agarose overnight. Ubiquitin, MDM2, and p53 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-231 cells were transfected with siRNAs for PIPKIα and treated with vehicle or 30 μM cisplatin for 24 h. Empty vector (Mock) was used as a negative control. After cells were treated with 10 μM of MG132 for 4 h, cells were harvested for IP of MDM2 and p53. Expression of the indicated proteins was analyzed by IB, and ubiquitin IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown; UT, untreated; Cis, cisplatin treated; Cis/MG132, Cisplatin/MG132 treated. D , MDA-MB-468 cells were transfected with siRNAs for αBC and HSP27 for 48 h in absence or presence of MG132. Empty vector (Mock) was used as a negative control. Cells were processed for IP of MDM2. IB was used to analyze indicated proteins, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown. E , a model of PIP 2 regulation of the interaction between MDM2 and sHSPs, which differentially control the stability of MDM2 and function of of MDM2. In the presence of PIPKIα, PIP 2 is linked to MDM2, which recruits αBC to MDM2 to stabilize it and enhances p53 binding. Conversely, in the absence of PIPKIα and PIP 2 , HSP27 is recruited to MDM2, which increases the ubiquitin E3 ligase activity of MDM2.

Techniques: Ubiquitin Proteomics, Incubation, In Vitro, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Control, Binding Assay, Activity Assay

HNE2 cells were transfected with a TP53 expression vector (the pCMV-p53 plasmid). Levels of ( A ) TP53 mRNA transcripts and ( B ) p53 protein expression were determined at 0–48 h post-transfection by qRT-PCR and western blotting, respectively. ( C ) To measure p53 transcriptional activity, the pCMV-p53 and the pp53-TA-luc plasmids were cotransfected into HNE2 cells, and transcriptional activity of p53 from 0–48 h post-transfection was determined by luciferase assays. Similarly, induction of ( D ) MDM2 mRNA transcripts and ( E ) MDM2 protein expression in HNE2 were also determined in HNE2 pCMV-p53 transfectants by qRT-PCR and western blotting. GAPDH protein expression was detected as a loading control for p53 and MDM2 western blots. Data shown are representative of 3 independent experiments. Bar graphs show mean ± S.D. ** P <0.01, *** P <0.001.

Journal: PLoS ONE

Article Title: LOC401317, a p53-Regulated Long Non-Coding RNA, Inhibits Cell Proliferation and Induces Apoptosis in the Nasopharyngeal Carcinoma Cell Line HNE2

doi: 10.1371/journal.pone.0110674

Figure Lengend Snippet: HNE2 cells were transfected with a TP53 expression vector (the pCMV-p53 plasmid). Levels of ( A ) TP53 mRNA transcripts and ( B ) p53 protein expression were determined at 0–48 h post-transfection by qRT-PCR and western blotting, respectively. ( C ) To measure p53 transcriptional activity, the pCMV-p53 and the pp53-TA-luc plasmids were cotransfected into HNE2 cells, and transcriptional activity of p53 from 0–48 h post-transfection was determined by luciferase assays. Similarly, induction of ( D ) MDM2 mRNA transcripts and ( E ) MDM2 protein expression in HNE2 were also determined in HNE2 pCMV-p53 transfectants by qRT-PCR and western blotting. GAPDH protein expression was detected as a loading control for p53 and MDM2 western blots. Data shown are representative of 3 independent experiments. Bar graphs show mean ± S.D. ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies used included the following: rabbit monoclonal antibodies against p53 (Cell Signaling Technology, Danvers, MA, USA), p21 (Cell Signaling Technology), cyclin D1 (Abcam, Cambridge, Massachusetts, USA), and α-tubulin (Abcam); mouse monoclonal antibodies against cyclin E1 (Cell Signaling Technology) and GAPDH (Cell Signaling Technology); rabbit polyclonal antibodies against MDM2 (Abcam), poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology), and cleaved caspase-3 (Cell Signaling Technology).

Techniques: Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Control

Def and CAPN3 duet in zebrafish. (A) WISH analysis of the expression patterns of capn3a and capn3b in embryos at 2 and 4 dpf, respectively, using capn3a or capn3b probes. en, endoderm tube; in, intestine; le, lens; lv, liver. (B) Western blot of p53, Def and Mdm2 for comparing the effect of knockdown of zebrafish Capn3a or Capn3b with their specific morpholinos capn3a-MO or capn3b-MO on Def-mediated or Mdm2-mediated p53 degradation in the injected embryos at 6 hpi. (C) Western blot of the endogenous p53 in capn3a -MO, capn3b -MO and def -MO morphants at 3 dpi. (D) Western blot of p53 for examining the effect of capn3b mRNA lacking the capn3b-MO target sequence ( Myc-capn3b-5mu ) or the mutant mRNA carrying a mutation changing the codon for the active site Cys 120 to Ser 120 ( Myc-capn3b C120S -5mu ) on elevated endogenous p53 induced by capn3b-MO . Capn3b was detected using a polyclonal antibody against zebrafish Capn3b. Myc-Capn3b, Myc-tagged Capn3b; endo-Capn3b, endogenous Capn3b. (E) qPCR analysis of capn3a and capn3b transcripts in def hi429 mutant embryos and wild-type controls (WT). The relative expression level of the genes was shown in fold change as normalized against zebrafish elf1 . (F) Western blot analysis of p53, Δ113p53, Def and CAPN3 (α-Myc) in the embryos co-injected with def -MO morphlino plus capn3a mRNA or def -MO morpholino plus capn3b mRNA at 3 dpi. Wild-type embryos (CK) and st-MO-injected embryos were used as the controls. (G) Graphics summarizes the roles of the nucleolus in regulation of p53 homeostasis. In response to stress conditions such as nucleolar disruption, oncogene activation, DNA damage or developmental defect, p53 is stabilized or activated. Nucleolar factors including Arf, PML, RPL5 and RPL11 can associate with Mdm2 to prevent p53 ubiquitination and degradation through the 26S proteasome in the nucleoplasm and cytoplasm. In contrast, the role of the Def-CAPN3 pathway is to prevent the accumulation of p53 in the nucleolus by triggering in situ p53 degradation.

Journal: Cell Research

Article Title: Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation

doi: 10.1038/cr.2013.16

Figure Lengend Snippet: Def and CAPN3 duet in zebrafish. (A) WISH analysis of the expression patterns of capn3a and capn3b in embryos at 2 and 4 dpf, respectively, using capn3a or capn3b probes. en, endoderm tube; in, intestine; le, lens; lv, liver. (B) Western blot of p53, Def and Mdm2 for comparing the effect of knockdown of zebrafish Capn3a or Capn3b with their specific morpholinos capn3a-MO or capn3b-MO on Def-mediated or Mdm2-mediated p53 degradation in the injected embryos at 6 hpi. (C) Western blot of the endogenous p53 in capn3a -MO, capn3b -MO and def -MO morphants at 3 dpi. (D) Western blot of p53 for examining the effect of capn3b mRNA lacking the capn3b-MO target sequence ( Myc-capn3b-5mu ) or the mutant mRNA carrying a mutation changing the codon for the active site Cys 120 to Ser 120 ( Myc-capn3b C120S -5mu ) on elevated endogenous p53 induced by capn3b-MO . Capn3b was detected using a polyclonal antibody against zebrafish Capn3b. Myc-Capn3b, Myc-tagged Capn3b; endo-Capn3b, endogenous Capn3b. (E) qPCR analysis of capn3a and capn3b transcripts in def hi429 mutant embryos and wild-type controls (WT). The relative expression level of the genes was shown in fold change as normalized against zebrafish elf1 . (F) Western blot analysis of p53, Δ113p53, Def and CAPN3 (α-Myc) in the embryos co-injected with def -MO morphlino plus capn3a mRNA or def -MO morpholino plus capn3b mRNA at 3 dpi. Wild-type embryos (CK) and st-MO-injected embryos were used as the controls. (G) Graphics summarizes the roles of the nucleolus in regulation of p53 homeostasis. In response to stress conditions such as nucleolar disruption, oncogene activation, DNA damage or developmental defect, p53 is stabilized or activated. Nucleolar factors including Arf, PML, RPL5 and RPL11 can associate with Mdm2 to prevent p53 ubiquitination and degradation through the 26S proteasome in the nucleoplasm and cytoplasm. In contrast, the role of the Def-CAPN3 pathway is to prevent the accumulation of p53 in the nucleolus by triggering in situ p53 degradation.

Article Snippet: Mouse monoclonal antibodies against human p53 (DO-1, sc-126), Hdm2 (SMP14, sc-965), p21 (F-5, sc-6246), C23 (H-6, sc-55486) and rabbit polyclonal antibody against human Bax (N-20, sc-493) were from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Knockdown, Injection, Sequencing, Mutagenesis, Disruption, Activation Assay, Ubiquitin Proteomics, In Situ

The transactivation and the phosphorylation of AR are disrupted by the XPD/R683W mutation. (A) GM03348D (XPD/WT), TTD12PV (XPD/R112H), XPJCLO (XPD/R683W) and TTD8PV (XPD/R722W) fibroblasts were transiently co-transfected with 100 ng of pGL3.PSA-Luc, pCMV.β-gal and either pSG5.AR (AR) or pSG5.RARα (RARα) or the corresponding empty vector. The cells were then treated with a specific ligand for AR (DHT, 10−7 M) or RARα (t-RA, 10−8 M). Luciferase activity was measured 24 h later and normalized to β-galactosidase activity. The results are the mean of three different experiments. (B) In vivo phosphorylation of AR was investigated in HeLa (WT), HD2 (XPD) and XPD cells overexpressing XPD/WT upon AR immunoprecipitation, autoradiography ([32P]-AR) and western blotting (AR). Quantitative analysis of AR phosphorylation in WT and XPD cells represents the ratio autoradiography/western blot signals ([32P]-AR/AR). (C) AR interacts with TFIIH. (Left panel) Endogenous AR was immunoprecipitated (IP:AR, lane 5) from LNCaP cells and after washes (150 mM NaCl), co-precipitated TFIIH was detected using an anti-XPB antibody. The control IP (IP neg, lane 4) was performed with a non-specific antibody. The input (lane 1) represents 10% of the total volume of LNCaP extract used for the immunoprecipitation. His-AR (lane 2) and purified XPB (lane 3) were used as size markers for the immunodetection of the endogeneous AR and XPB in LNCaP cells. (Right panel) Highly purified-recombinant AR was incubated with Sf9 cell extracts overexpressing TFIIH complex containing a FLAG-tagged p34, in the presence or absence of DHT (10−6 M). Immunoprecipitation was performed using an anti-FLAG antibody (IP:IIH) and after extensive washes (300 mM NaCl) and SDS–PAGE, bound proteins were detected with an anti-AR, XPD, p62, p44 and cdk7 antibodies. (D) Equal amounts of highly purified-recombinant AR was incubated with Sf9 cell extracts overexpressing separately the subunit XPB, XPD, p44 or cdk7 of TFIIH in the presence or absence of DHT (10−6 M). After AR immunoprecipitation, co-precipitated TFIIH subunits were detected by western blot. The control IP (lane 1) was performed without AR. (E) AR, A/B.AR, ARΔA/B, PPARα or A/B.AR S515A were incubated with purified-recombinant TFIIH in the presence or absence of DHT (10−6 M) as indicated. After SDS–PAGE, each protein was resolved by Coomassie blue staining (Stain.) and radioactive labelling was analysed by autoradiography (Auto.). The complete Coomassie blue stainings and western blots of the different purified-recombinant proteins are shown in Supplementary Figure S1A. (F) WT (HeLa) and XPD (HD2) cells were transiently co-transfected with 100 ng of pGL3.PSA-Luc, pCMV.β-gal and pSG5 AR/WT, /S515A, or /S515E before DHT (10−7 M) treatment. The results were obtained as described in A.

Journal: The EMBO Journal

Article Title: The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process

doi: 10.1038/emboj.2010.337

Figure Lengend Snippet: The transactivation and the phosphorylation of AR are disrupted by the XPD/R683W mutation. (A) GM03348D (XPD/WT), TTD12PV (XPD/R112H), XPJCLO (XPD/R683W) and TTD8PV (XPD/R722W) fibroblasts were transiently co-transfected with 100 ng of pGL3.PSA-Luc, pCMV.β-gal and either pSG5.AR (AR) or pSG5.RARα (RARα) or the corresponding empty vector. The cells were then treated with a specific ligand for AR (DHT, 10−7 M) or RARα (t-RA, 10−8 M). Luciferase activity was measured 24 h later and normalized to β-galactosidase activity. The results are the mean of three different experiments. (B) In vivo phosphorylation of AR was investigated in HeLa (WT), HD2 (XPD) and XPD cells overexpressing XPD/WT upon AR immunoprecipitation, autoradiography ([32P]-AR) and western blotting (AR). Quantitative analysis of AR phosphorylation in WT and XPD cells represents the ratio autoradiography/western blot signals ([32P]-AR/AR). (C) AR interacts with TFIIH. (Left panel) Endogenous AR was immunoprecipitated (IP:AR, lane 5) from LNCaP cells and after washes (150 mM NaCl), co-precipitated TFIIH was detected using an anti-XPB antibody. The control IP (IP neg, lane 4) was performed with a non-specific antibody. The input (lane 1) represents 10% of the total volume of LNCaP extract used for the immunoprecipitation. His-AR (lane 2) and purified XPB (lane 3) were used as size markers for the immunodetection of the endogeneous AR and XPB in LNCaP cells. (Right panel) Highly purified-recombinant AR was incubated with Sf9 cell extracts overexpressing TFIIH complex containing a FLAG-tagged p34, in the presence or absence of DHT (10−6 M). Immunoprecipitation was performed using an anti-FLAG antibody (IP:IIH) and after extensive washes (300 mM NaCl) and SDS–PAGE, bound proteins were detected with an anti-AR, XPD, p62, p44 and cdk7 antibodies. (D) Equal amounts of highly purified-recombinant AR was incubated with Sf9 cell extracts overexpressing separately the subunit XPB, XPD, p44 or cdk7 of TFIIH in the presence or absence of DHT (10−6 M). After AR immunoprecipitation, co-precipitated TFIIH subunits were detected by western blot. The control IP (lane 1) was performed without AR. (E) AR, A/B.AR, ARΔA/B, PPARα or A/B.AR S515A were incubated with purified-recombinant TFIIH in the presence or absence of DHT (10−6 M) as indicated. After SDS–PAGE, each protein was resolved by Coomassie blue staining (Stain.) and radioactive labelling was analysed by autoradiography (Auto.). The complete Coomassie blue stainings and western blots of the different purified-recombinant proteins are shown in Supplementary Figure S1A. (F) WT (HeLa) and XPD (HD2) cells were transiently co-transfected with 100 ng of pGL3.PSA-Luc, pCMV.β-gal and pSG5 AR/WT, /S515A, or /S515E before DHT (10−7 M) treatment. The results were obtained as described in A.

Article Snippet: Polyclonal antibodies against TFIIH subunit cdk7 (C-19), AR (C-19), CHIP (H-231), Ubiquitin (FL-76), MDM2 (SMP14), β-tubulin and 20S proteasome β5 (C-19) were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Plasmid Preparation, Luciferase, Activity Assay, In Vivo, Immunoprecipitation, Autoradiography, Western Blot, Control, Purification, Immunodetection, Recombinant, Incubation, SDS Page, Staining

$AR phosphorylation status selectively promotes the recruitment of ubiquitin–proteasome components at the PSA promoter. WT and XPD cells were transiently transfected to overexpress either AR/WT, AR/WT together with XPD/WT or AR/S515E (as indicated at the top of each panel). (A–D) Expression of the PSA gene: RT–qPCR analysis was performed at indicated times after DHT (10−7 M) treatment. The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n-fold induction relative to non-treated cells. (E–H) After DHT treatment, the recruitment of RNA pol II (yellow curve), TFIIH (via its XPB and cdk7 subunits, blue and green curve, respectively) and AR (red curve) were analysed by ChIP assays at the PSA proximal promoter. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). (I–L) Recruitment of the MDM2 (green curve) and CHIP (yellow curve) E3 ligases on the PSA promoter. The recruitment of the ubiquitinated AR-containing fraction (brown curve) was also analysed by ChIP/re-ChIP assays using first an anti-ubiquitin antibody and second with an anti-AR antibody. (M–P) Recruitment of SUG1 (red curve), β5 (light blue) and S1 (dark blue) subunits of the proteasome at the PSA promoter was analysed by ChIP assays. Note that the recruitment of S1 has not been analysed in XPD cells transiently co-transfected with plasmids encoding AR/WT and XPD/WT (O).$

Journal: The EMBO Journal

Article Title: The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process

doi: 10.1038/emboj.2010.337

Figure Lengend Snippet: AR phosphorylation status selectively promotes the recruitment of ubiquitin–proteasome components at the PSA promoter. WT and XPD cells were transiently transfected to overexpress either AR/WT, AR/WT together with XPD/WT or AR/S515E (as indicated at the top of each panel). (A–D) Expression of the PSA gene: RT–qPCR analysis was performed at indicated times after DHT (10−7 M) treatment. The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n-fold induction relative to non-treated cells. (E–H) After DHT treatment, the recruitment of RNA pol II (yellow curve), TFIIH (via its XPB and cdk7 subunits, blue and green curve, respectively) and AR (red curve) were analysed by ChIP assays at the PSA proximal promoter. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). (I–L) Recruitment of the MDM2 (green curve) and CHIP (yellow curve) E3 ligases on the PSA promoter. The recruitment of the ubiquitinated AR-containing fraction (brown curve) was also analysed by ChIP/re-ChIP assays using first an anti-ubiquitin antibody and second with an anti-AR antibody. (M–P) Recruitment of SUG1 (red curve), β5 (light blue) and S1 (dark blue) subunits of the proteasome at the PSA promoter was analysed by ChIP assays. Note that the recruitment of S1 has not been analysed in XPD cells transiently co-transfected with plasmids encoding AR/WT and XPD/WT (O).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Expressing, Quantitative RT-PCR, Immunoprecipitation

Silencing either cdk7 or MDM2 promotes CHIP recruitment at the PSA promoter. WT cells stably expressing AR/WT were transfected with either a pool of non-targeting si-RNAs (used as control, si-ctl, panels A, E, I, M, Q), si-cdk7 (panels B, F, J, N, R), si-MDM2 (panels C, G, K, O) or si-CHIP (panels D, H, L, P). (A–D) PSA gene expression was analysed by RT–qPCR at indicated times after DHT (10−7 M) treatment in cells transfected with si-ctl (A), si-cdk7 (B), si-MDM2 (C) or si-CHIP (D). The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n-fold induction relative to non-treated cells. (E–H) The recruitment of RNA pol II (yellow bars) and AR (orange bars) at the PSA promoter was analysed by ChIP assays. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). (I–L) Recruitment of the E3 ligases MDM2 (green bars) and CHIP (yellow bars) at the PSA promoter was next investigated. (M–P) ChIP assays analysed the recruitment of the proteasomal subunits β5 (green bars), S1 (blue bars) and SUG1 (red bars). (Q–R) [35S] Pulse chase of AR protein in cells stably expressing AR/WT and transfected with either si-ctl (Q) or si-cdk7 (R). Experiments were performed as described in Figure 2. The values are the mean±s.e.m. of three independent experiments.

Journal: The EMBO Journal

Article Title: The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process

doi: 10.1038/emboj.2010.337

Figure Lengend Snippet: Silencing either cdk7 or MDM2 promotes CHIP recruitment at the PSA promoter. WT cells stably expressing AR/WT were transfected with either a pool of non-targeting si-RNAs (used as control, si-ctl, panels A, E, I, M, Q), si-cdk7 (panels B, F, J, N, R), si-MDM2 (panels C, G, K, O) or si-CHIP (panels D, H, L, P). (A–D) PSA gene expression was analysed by RT–qPCR at indicated times after DHT (10−7 M) treatment in cells transfected with si-ctl (A), si-cdk7 (B), si-MDM2 (C) or si-CHIP (D). The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n-fold induction relative to non-treated cells. (E–H) The recruitment of RNA pol II (yellow bars) and AR (orange bars) at the PSA promoter was analysed by ChIP assays. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). (I–L) Recruitment of the E3 ligases MDM2 (green bars) and CHIP (yellow bars) at the PSA promoter was next investigated. (M–P) ChIP assays analysed the recruitment of the proteasomal subunits β5 (green bars), S1 (blue bars) and SUG1 (red bars). (Q–R) [35S] Pulse chase of AR protein in cells stably expressing AR/WT and transfected with either si-ctl (Q) or si-cdk7 (R). Experiments were performed as described in Figure 2. The values are the mean±s.e.m. of three independent experiments.

Techniques: Stable Transfection, Expressing, Transfection, Control, Gene Expression, Quantitative RT-PCR, Immunoprecipitation, Pulse Chase

TFIIH-mediated phosphorylation of AR regulates its turnover by triggering its degradation by the ubiquitin/proteasome pathway. Upon the DHT ligand induction, the transactivation complex is formed once AR homodimer has targeted its responsive element (ARE) at the PSA promoter; co-factors are assembled at the promoter together with RNA pol II and the general transcription factors (GTFs) including TFIIH. In WT cells, the AR/S515 phosphorylation by TFIIH (via its cdk7 subunit) promotes the recruitment of both MDM2 E3 ligase that helps for AR polyubiquitination and the proteasome. In XPD cells (bearing the XPD/R683W mutation), AR/S515 phosphorylation is strongly inhibited, preventing the recruitment of MDM2. The E3 ligase CHIP is thus preferentially recruited, which allows with a lesser efficiency the AR polyubiquitin/proteasome process resulting in a much slower turnover.

Journal: The EMBO Journal

Article Title: The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process

doi: 10.1038/emboj.2010.337

Figure Lengend Snippet: TFIIH-mediated phosphorylation of AR regulates its turnover by triggering its degradation by the ubiquitin/proteasome pathway. Upon the DHT ligand induction, the transactivation complex is formed once AR homodimer has targeted its responsive element (ARE) at the PSA promoter; co-factors are assembled at the promoter together with RNA pol II and the general transcription factors (GTFs) including TFIIH. In WT cells, the AR/S515 phosphorylation by TFIIH (via its cdk7 subunit) promotes the recruitment of both MDM2 E3 ligase that helps for AR polyubiquitination and the proteasome. In XPD cells (bearing the XPD/R683W mutation), AR/S515 phosphorylation is strongly inhibited, preventing the recruitment of MDM2. The E3 ligase CHIP is thus preferentially recruited, which allows with a lesser efficiency the AR polyubiquitin/proteasome process resulting in a much slower turnover.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis

Review

polyclonal antibodies against mdm2 (Cell Signaling Technology Inc)

Structured Review

Images

1) Product Images from "Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex"

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