Journal: Oncotarget

Article Title: AKT regulates NPM dependent ARF localization and p53mut stability in tumors.

doi: 10.18632/oncotarget.2178

Figure Lengend Snippet: Fig.4: Inhibition of AKT promotes enhanced MDM2 activity via the increased association between NPM and p14ARF. (A) Npm-/-, p53-/-double null MEF were infected with pBABE retrovirus empty vector and pBABE expressing FLAG-tagged-NPM-WT, NPM-S48A or S48E as indicated. Immunopurification of NPM was done by pulling down with the Flag tag (middle panel) followed by elution of complexes by the Flag peptide and subsequent immunopurification of endogenous MDM2 (lower panel). (B) Nuclear immunoprecipitates of MDM2 from T24 cells treated with MK-2206 (5 μM, 24 hrs). Immunoprecipitates and lysates were blotted with the indicated antibodies. (C) T24 cells were treated with MK-2206 (5 μM) as indicated. p14ARF was immunoprecipitated from whole cell lysates and nuclear extracts and the association with NPM and MDM2 determined by western blot. Immunoprecipitates and lysates were blotted with the indicated antibodies. (D) MDM2 and (E) p53 ubiquinitation assay in H1299 cells transfected with wild type p53, HA-tagged ubiquitin and treated for 16 hrs with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) as indicated. Immunoprecipitates and whole cell lysates were probed with the indicated antibodies.

Article Snippet: The following plasmids were purchased from Addgene; pBABE puro-myr-FLAGAKT1 (Addgene plasmid 15294), [104], pBABE PuroL myr-HAAKT2 (Addgene plasmid 9018), pBABE-puro-KRas V12 (Addgene plasmid 9052) and pcDNA3 MDM2 S166D S186D (Addgene plasmid 16236).

Techniques: Inhibition, Activity Assay, Infection, Plasmid Preparation, Expressing, Immu-Puri, FLAG-tag, Immunoprecipitation, Western Blot, Transfection, Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The ubiquitination of GRK2 is involved in D2R β-arrestin pathway-mediated ERK activation. ( A ) CTRL-KD and Mdm2-KD cells were transfected with plasmids encoding [Gprot] D2R or [βarr] D2R. Serum-starved cells were stimulated with 10 μM DA for 2 min ( [Gprot] D2R producing) or 10 min ( [βarr] D2R producing). CTRL-KD and Mdm2-KD cell lysates were immunoblotted using p-ERK1/2 (1:1000 dilution) and ERK2 (1:1000 dilution) antibodies, respectively. Cell lysates were immunoblotted with Mdm2 or β-actin antibodies. About 86% of the Mdm2 levels in cells were diminished. ** p < 0.01 compared with the corresponding Veh group, # p < 0.05 compared with the DA stimulation group ( n = 3). ( B ) GRK2-KD cells were transfected with plasmids encoding [Gprot] D2R or [βarr] D2R, and co-transfected with plasmids encoding GRK2-WT or GRK2-4KR. Serum-starved cells were treated with 10 μM DA for 2 min ( [Gprot] D2R-producing) or 10 min ( [βarr] D2R-producing). Cells lysates were immunoblotted using p-ERK1/2 (1:1000 dilution) and ERK2 (1:1000 dilution) antibodies, respectively. We measured the levels of p-ERKs and total ERKs in the same sample and then divided the amount of pERKs by the amount of total ERKs to obtain the p-ERK/ERK ratio. The pERK/ERK ratio provided a normalized measure of ERK pathway activation. ** p < 0.01, * p < 0.05 compared with the corresponding Veh group, ## p < 0.01 compared with the DA/GRK2-WT/ [βarr] D2R expression group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Activation Assay, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The activation of the D2R β-arrestin pathway promotes GRK2 ubiquitination. ( A ) HEK 293 cells producing [Gprot] D2R or [βarr] D2R were transfected with plasmids encoding GFP-GRK2 and FLAG-Mdm2. The cells were stimulated with either Veh or 10 μM DA for 2 min. FLAG beads were used to immunoprecipitate cell lysates. Co-IP/Lysate and IP were immunoblotted via the use of GFP (1:1000 dilution) and FLAG (1:1000 dilution) antibodies, respectively. The data represent the outcomes of three independent investigations with comparable results. ** p < 0.01 compared with the Veh group ( n = 3). ( B ) HEK 293 cells were transfected with plasmids encoding [Gprot] D2R or [βarr] D2R, HA-Ub, and FLAG-GRK2. The cells were treated for 2 min with either a vehicle or 10 μM DA. Immunoprecipitation of cell lysates using FLAG beads. Antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution) were used to immunoblot Co-IP and IP. ** p < 0.01 compared with the Veh group ( n = 3). ( C ) HEK 293 cells producing D2R were transfected with plasmids encoding HA-Ub and FLAG-GRK2. The cells were stimulated with either 10 μM MLS1547 or 1 μM UNC9994 for 2 min. Immunoprecipitation of cell lysates using FLAG beads. Antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution) were used to immunoblot Co-IP and IP. * p < 0.05 compared with the Veh group ( n = 3). ( D ) HEK 293 cells were transfected with plasmids encoding D2R and FLAG-GRK2. Cells were prestimulated with 50 μg/mL cycloheximide for 1 h, followed by 10 μM MLS1547 or 1 μM UNC9994 treatment for 0–2 h. Cell lysates were immunoblotted using FLAG (1:1000 dilution) or β-actin (1:2000 dilution) antibodies. “0 min” groups were normalized to 100%. * p < 0.05, *** p < 0.001 compared with the Veh group ( n = 3). ( E ) CTRL-KD and Mdm2-KD cells producing D2R were transfected with plasmids encoding GRK2. The pretreatment of cells with 50 μg/mL cycloheximide for 1 h was followed with treatment with 1 μM UNC9994 for 0–2 h. The immunoblotting of cell lysates with antibodies against GRK2 (1:2000 dilution) or β-actin (1:2000 dilution) was performed. “0 min” groups were normalized to 100%. * p < 0.05, *** p < 0.001 compared with the Veh/CTRL-KD group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The Mdm2-mediated ubiquitination of GRK2 occurs in the cytoplasm in response to UNC9994 stimulation. ( A ) HEK 293 cells were transfected with plasmids encoding GFP-GRK2 or GFP-β-arrestin2. Cells were exposed to a vehicle or 10 ng/mL LMB for 3 h. The horizontal bar represents 10 μm. One representative example from three independent investigations is depicted in the data. ( B ) HEK 293 cells were transfected with plasmids encoding FLAG-D2R and GFP-Mdm2. Cells were stimulated with either a vehicle or 1 μM UNC9994 for 10 min. Later, the cells were incubated with FLAG antibodies (1:1000 dilution) and Alexa-594-conjugated anti-rabbit secondary antibodies (1:500 dilution) in succession. The horizontal bar represents 10 μm. One representative example from three independent investigations is depicted in the data. ( C ) HEK 293 cells were transfected with plasmids encoding D2R, HA-Ub, and FLAG-GRK2. Cells were pretreated with 10 ng/mL LMB for 3 h, followed by 1 μM UNC9994 treatment for 2 min. Cell lysates were immunoprecipitated via the use of FLAG beads. Co-IP and IP were immunoblotted with HA (1:1000 dilution) and FLAG (1:1000 dilution) antibodies, respectively. *** p < 0.001 compared with the Veh/veh group ( n = 3). ( D ) HEK 293 cells were transfected with plasmids encoding D2R. Cells were stimulated with 1 μM UNC9994 for 2 min. The fractionation of cell lysates followed the protocol outlined in the “ ”. Nuclear and cytosolic fractions were utilized in ubiquitination assays. NF, nuclear fraction; CF, cytosolic fraction. ** p < 0.01 compared with the Veh/CF group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay, Fractionation

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The tyrosine phosphorylation of GRK2 is required for Mdm2-mediated GKR2 ubiquitination upon the stimulation of the D2R β-arrestin-dependent pathway. ( A ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, GRK2, and FLAG-Mdm2. The pretreatment of cells with 10 μM PP2 for 30 min was followed by a 2-min treatment with 10 μM DA. The immunoprecipitation of cell lysates using FLAG beads. Co-IP/Lysate and IP were immunoblotted, respectively, with antibodies against GRK2 (1:2000) and FLAG (1:1000). ( B ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, HA-Ub, and FLAG-GRK2. Cells were pretreated with 10 μM PP2 for 30 min, followed by 10 μM DA treatment for 2 min. The immunoprecipitation of cell lysates using FLAG beads. Co-IP/Lysate and IP were immunoblotted, respectively, with antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution). ** p < 0.01 compared with the Veh group ( n = 3). ( C ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, HA-Ub, and FLAG-GRK2-WT or FLAG-GRK2-3YF. The cells were treated with either a vehicle or 10 μM DA for 2 min. Immunoprecipitation of cell lysates using FLAG beads. Co-IP/Lysate and IP were immunoblotted with antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution), respectively. ** p < 0.01 compared with the Veh group ( n = 3). ( D ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, HA-Src, and FLAG-GRK2-WT or FLAG-GRK2-4KR. Cells were treated with either a vehicle or 10 μM DA for 2 min. The immunoprecipitation of cell lysates using FLAG beads. HA (1:1000 dilution) and FLAG (1:1000 dilution) antibodies were used to immunoblot Co-IP/Lysate and IP, respectively. ** p < 0.01, *** p < 0.001 compared with corresponding Veh group (n = 3). ( E ) GRK2-KD cells producing [βarr] D 2 R were transfected with plasmids encoding GRK2-WT or GRK2-3YF. Serum-starved cells were incubated in the presence of 10 μM DA for 10 min. Antibodies against p-ERK1/2 (1:1000 dilution) and ERK2 (1:1000 dilution) were used to immunoblot lysates. ** p < 0.01 compared with the corresponding Veh/WT group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: Diagram showing the mechanisms involved in D2R β-arrestin-dependent pathway-mediated ERK activation. After stimulation with an agonist to activate the D2R β-arrestin signaling pathway, Mdm2 moves out of the nucleus to ubiquitinate GRK2, which is in an Src-dependent tyrosine phosphorylation state. Ubiquitinated GRK2 then translocates to the plasma membrane and interacts with activated D2R, followed by the phosphorylation of D2R and recruiting β-arrestin to mediate downstream ERK signal transduction.

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Transduction

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 1. Matrine inhibits the expression of MDM2 in Hep3B and L0‑2 cells. Western blotting was performed to determine (A) the protein expression levels of MDM2 following treatment with 4.0 mg/ml matrine for 24 h, (B) the dose‑response of MDM2 inhibition following treatment with the indicated concentrations of matrine for 24 h, (C) the time‑response of MDM2 inhibition following treatment with 0.5 mg/ml matrine for the indicated durations and (D) the protein expression of MDM2 following treatment with 5 Gy IR for the indicated durations. Western blotting was performed for both in Hep3B and L0‑2 cells, and β‑actin was used as a loading control. Con, control; MDM, mouse double‑minute; IR, irradiation.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Inhibition, Control, Irradiation

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 2. Examination of the molecular mechanism by which matrine reduces MDM2. (A) The mRNA expression of MDM2 in Hep3B cells was determined by RT‑qPCR following treatment with 0.5 mg/ml matrine for the indicated durations. The data is shown as the fold change. (B) Hep3B cells were pre‑treated with or without 10 µM CHX for 10 min, followed by incubation with or without 0.5 mg/ml matrine for 4 h, then 5 mg/ml actino mycin D was added. Cells were then harvested and the mRNA expression levels of MDM2 were determined by RT‑qPCR. The data are presented as the mean ± standard deviation (*P<0.05 and **P<0.01 compared with the untreated 0 h samples). (C) A CHX pulse‑chase assay was performed to detect MDM2 turnover in Hep3B cells treated with or without 0.5 mg/ml matrine for 4 h. The numerical labels under each protein band represents the expression levels after normalization against β‑actin, compared with untreated samples (defined as 1 unit). MDM, mouse double‑minute; CHX, cycloheximide; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Standard Deviation, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 3. Matrine‑induced downregulation of MDM2 results in an increase in the expression of p73α. (A) Hep3B cells were treated with different concentra tions of either benzo(a)pyrene (16, 32 or 64 µM) or matrine (1.0, 2.0 or 4.0 mg/ml) for 12 h. The mRNA expression levels of p53 were assessed by reverse transcription‑quantitative polymerase chain reaction. The data are presented as the mean ± standard deviation (**P<0.01 compared with the untreated 0 h group) samples. (B) Hep3B cells were pre‑treated with or without 10 µM CHX for 10 min, followed by an incubation with 0.5 mg/ml matrine. The cell extracts from 0 or 2 or 4 h treatment groups were assessed by western blotting to determine the expression of p73α. (C) A pulse‑chase assay for p73α turnover was performed in Hep3B cells treated with 0.5 mg/ml matrine. (D) Hep3B cells treated with 4.0 mg/ml matrine for the indicated durations were assessed for the expression levels of MDM2 and p73α by western blotting. β‑actin was used as a loading control. CHX, cycloheximide; MDM, mouse double‑minute.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Standard Deviation, Incubation, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 4. Matrine‑increased expression of p73α causes no induction of its targets, p21 and PUMA, as compared with treatment with Benzo(a)pyrene or epoto side. (A) Hep3B cells were treated with 32 µM Benzo(a)pyrene or 0.5 mg/ml matrine for 12 h and subsequently, the mRNA expression levels of p73α, MDM2, p21 and PUMA were assessed by reverse transcription‑quantitative polymerase chain reaction. The data are presented as the fold change compared with the control group. (B) Hep3B cells were treated with either 100 µM epotoside or 0.5 mg/ml matrine for various durations. The protein expression levels of p73α, MDM2, p21 and PUMA were assessed by western blotting. β‑actin was used as a loading control. (C) Cell‑cycle analysis in Hep3B cells was performed 4 h post‑treatment with 100 µM epotoside or 0.5 mg/ml matrine. MDM, mouse double‑minute; PUMA, p53 upregulated modulator of apoptosis.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Control, Western Blot, Cell Cycle Assay

Journal: Molecular medicine reports

Article Title: Matrine‑induced apoptosis in Hep3B cells via the inhibition of MDM2.

doi: 10.3892/mmr.2016.5999

Figure Lengend Snippet: Figure 5. Effect of epotoside‑increased and matrine‑decreased MDM2 on the expression of IAP3. (A) Hep3B cells were incubated with either 100 µM epo toside or 0.5 mg/ml matrine for the indicated duration. The protein expression levels of MDM2 and IAP3 were assessed by western blotting. (B) Normal and MDM2 overexpression Hep3B cells were treated with 0.5 mg/ml matrine for the indicated durations. The transfection efficiency of MDM2 and IAP3 in MDM2‑overexpressing Hep3B cells were evaluated by western blotting. (C) The protein stability of IAP3 was detected using a pulse‑chase assay following treatment with 0.5 mg/ml matrine. β‑actin was used as a loading control. MDM, mouse double‑minute; IAP, inhibitor of apoptosis protein; CHX, cycloheximide.

Article Snippet: MDM2 CRISPR activation plasmid, and MDM2 (cat. no. BS1223), PUMA (cat. no. AB10418), p73α (cat. no. 4662), p73α (Try99) (cat. no. 4665S) and β-actin (cat. no. 4967) antibodies were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Over Expression, Transfection, Control