Journal: Oncoscience

Article Title: Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma

doi:

Figure Lengend Snippet: (A) Circos diagram depicting a chromothriptic event that occurred in TCGA-06-0157 which resulted in the recombination of discrete regions of chromosomes 1, 7, and 12, leading to the co-amplification of MDM4, EGFR, and CDK4, respectively. Tracks are organized, from outside to inside: karyotype data, cosmic database cancer associated genes, intra-chromosomal translocations (light green links), copy number data (0>cn<10, >10 is indicated by size of point), and inter-chromosomal translocation (central links). (B) cBioPortal OncoPrint visualization of TCGA Glioblastoma tumor alterations showing the mutual exclusivity of MDM2, MDM4, and TP53 paired with CDK4, RB1, and to a lesser extent, CDKN2A. (C) Specific TCGA tumors analyzed by this study were selected from the population of MDM2/MDM4 amplified tumors depicted in figure and include the tyrosine kinase receptors that are amplified. A more detailed table of these alterations is included in figure .

Article Snippet: Copy number analysis was performed on a Raindance Technologies Raindrop platform paired with Taqman probes for MDM2(ABI #Hs02370754_cn, EGFR (ABI #Hs04960197_cn), and RNaseP (ABI #4403326) using standard procedures outlined in their product manual.

Techniques: Amplification, Translocation Assay

Journal: Oncoscience

Article Title: Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma

doi:

Figure Lengend Snippet: (A) Circos diagram depicting all suspect chromothriptic regions resulting from Shatterproof analysis. (B) Enhancement of the chromosome 7 and 12 recombination seen in 3a supporting the assertion that EGFR , along with MDM2 and CDK4 , have been translocated onto the same amplicon. (C) FISH performed on paraffin embedded tissue from TCGA-19-2624 reveals that both EGFR and MDM2 are present at numerous loci within the nucleus, consistent with the formation of double minutes.

Article Snippet: Copy number analysis was performed on a Raindance Technologies Raindrop platform paired with Taqman probes for MDM2(ABI #Hs02370754_cn, EGFR (ABI #Hs04960197_cn), and RNaseP (ABI #4403326) using standard procedures outlined in their product manual.

Techniques: Amplification

Journal: Oncoscience

Article Title: Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma

doi:

Figure Lengend Snippet: (A) Quantitative digital PCR of MDM2 using RNAseP as a control indicates a copy number of 88. (B) Metaphase FISH performed on the 10-48 glioblastoma derived cell line reveals a homogeneously stained derivative chromosome containing many copies of MDM2 . (C) Metaphase FISH probing for 12p (green) and 12q (orange) telomeres indicates that these telomeres are intact and that fusion of this chromosome is not responsible for the formation of the derivative chromosome, both copies of which can be clearly distinguished based on their relative size. Note that the probe mix that was used additionally contained CEP 18 (aqua) and 18q (yellow). (D) 10-48 cells are highly sensitive to MDM2 inhibition. Clonogenic survival of 10-48 cells treated with 1μM or 10μM nutlin for 24 hours. Glioma cell lines U251 (lack functional P53) and U87 (wild type P53) are included as controls. * p <0.05. ** p <0.001. Significance determined by ANOVA. n=4.

Article Snippet: Copy number analysis was performed on a Raindance Technologies Raindrop platform paired with Taqman probes for MDM2(ABI #Hs02370754_cn, EGFR (ABI #Hs04960197_cn), and RNaseP (ABI #4403326) using standard procedures outlined in their product manual.

Techniques: Digital PCR, Control, Derivative Assay, Staining, Inhibition, Functional Assay

Journal: Scientific reports

Article Title: Tau protein binds to the P53 E3 ubiquitin ligase MDM2.

doi: 10.1038/s41598-023-37046-8

Figure Lengend Snippet: Figure 2. Detection of the endogenous Tau-MDM2 complex in cells and brain. (A) Hela cells expressing Tau2N4R-HAT10 and T11β1-MDM2 were treated with MG132 for 4 h before lysis and analysis by AlphaLisa to detect the Tau-MDM2 complex as well as the total amount of Tau or MDM2. Values are given as mean fold over negative control omitting one of the two primary antibodies (ctrl), n = 3, ± SD, unpaired two-tailed student t-test. (B) SH-SY5Y treated with MG132 were lysed and analyzed by AlphaLisa. Values are given as mean fold over negative control obtained from Tau-KO cells for Tau-MDM2 and Tau (KO) or also omitting the antibody for MDM2 (ctrl), n = 3, ± SD, unpaired two-tailed student t-test, or ordinary one-way ANOVA and Tukey’s multiple comparison test. (C) Identical quantities of the same SH-SY5Y cell lysate were subjected to affinity purification on the pDIQ peptide coupled to beads or on the same volume of uncoupled beads(ctrl). Cell lysates and affinity isolated proteins were resolved on a single gel and analyzed by western blot sequentially with the mouse Tau13-AF680 antibody and with the rabbit D1V2Z antibody and anti-rabbit IgG IRDye 800CW. Molecular weight markers are given on the left of the blots. Original blots are presented in supplementary material (Raw WB Sola). (D) Human brain homogenates from ten donors were subjected to AlphaLisa to detect the total amount of endogenous Tau-MDM2 complex as well as endogenous Tau or MDM2. Values are given as mean fold over negative control obtained by omitting one of the two primary antibodies (ctrl) n = 10, ± sem, unpaired two-tailed student t-test.

Article Snippet: After 10% SDS PAGE, PVDF membranes with transferred proteins were incubated with the primary antibodies indicated in the figures: 0.2 μg/mL Tau13-AlexaFluor (sc-21796 AF680, Santa Cruz), 0.2 μg/mL TauAS rabbit antiserum (ab64193, Abcam), 0.1 μg/mL rabbit monoclonal D1V2Z (86934, Cell Signaling), 0.1 μg/mL FL-393 rabbit antiserum, 0.4 μg/mL DO-1 antibody (sc-126, Santa Cruz) or 2.3 μg/mL β1 antibody.

Techniques: Expressing, Lysis, Negative Control, Two Tailed Test, Comparison, Affinity Purification, Isolation, Western Blot, Molecular Weight

Journal: Frontiers in Immunology

Article Title: Targeting hypoxia-induced tumor stemness by activating pathogen-induced stem cell niche defense

doi: 10.3389/fimmu.2022.933329

Figure Lengend Snippet: Bystander apoptosis is characterized by the HMGB1/p53 complex–mediated apoptosis. (A) p53 uptake by the EpCAM+/ABCG2+ versus EpCAM+/ABCG2- CSCs with or without anti-HMGB1 pre-treated BCG-CM. (B) Relative uptake of p53 by the EpCAM+/ABCG2+ and EpCAM+/ ABCG2- CSCs with or without neutralizing anti-HMGB1 pre-treated BCG-CM. (C) The induction of p53/MDM2 oscillation in EpCAM+/ABCG2+ CSCs following 12 h of BCG-CM treatment. (D) Significant induction of p53-related pro-apoptotic genes and HMGB1 gene in EpCAM+/ABCG2+ CSCs following 28 h of BCG-CM treatment. The real-time PCR data were compared with untreated EpCAM+/ABCG2+ CSCs to obtain fold change. (E) Significant increase in cleaved caspase-3 protein level in EpCAM+/ABCG2+ CSCs treated with BCG-CM with or without pifithrin alpha (2 µM in DMSO for 48 h) or anti-HMGB1 (10 µg/ml for 48 h; isotype control of same dose). Data represent means ± SEM (A–E) . N = 3 independent experiments (A–E) . *p < 0.05, **p < 0.01, and ***p < 0.001 ( A, B, E : one-way ANOVA with Dunnet post hoc test; C, D : Student’s t-test).

Article Snippet: The following TaqMan gene expression primers were used: human: ABCG2 (Hs00184979_m1), TLR2 (Hs02621280_s1), TLR4 (Hs00152939_m1), TLR7 (Hs01933259_s1), TLR9 (Hs00370913_s1), p53 (Hs01034249_m1), p21 (Hs00355782_m1), PUMA (Hs00248075_m1), Bax (Hs00180269_m1), GAPDH (Hs00266705_g1), MDM2 (Hs01066930_m1), and HMGB1 (Hs01923466_g1).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: Targeting hypoxia-induced tumor stemness by activating pathogen-induced stem cell niche defense

doi: 10.3389/fimmu.2022.933329

Figure Lengend Snippet: Tumor stemness defense (TSD) phenotype can amplify the pathogen-induced bystander apoptosis (PIBA). (A) The p53 uptake assay in the culture supernatant was measured from 0 to 16 h of BCG-CM treatment in the cells. The SCC-25 SP cells were obtained as described in Figure 1 Data represent mean +/- SEM, n= 3 independent experiments. ***p < 0.0001, student t test. (B) Potential mechanism of TSD phenotype–mediated niche defense of CSCs against BCG infection. In the infected CSCs, as part of the Altruistic stemness–based niche defense mechanism ( , , ) HMGB1 form a complex with cytoplasmic p53 to make an, “altruistic death signal”. TLR4 internalizes the altruistic death signal, leading to induction of p53/MDM2 oscillation and activation of p53-induced pro-apoptotic genes. The EpCAM+/ABCG2+ CSCs undergoing bystander apoptosis further release the HMGB1/p53 death complex into the TME, amplifying PIBA.

Article Snippet: The following TaqMan gene expression primers were used: human: ABCG2 (Hs00184979_m1), TLR2 (Hs02621280_s1), TLR4 (Hs00152939_m1), TLR7 (Hs01933259_s1), TLR9 (Hs00370913_s1), p53 (Hs01034249_m1), p21 (Hs00355782_m1), PUMA (Hs00248075_m1), Bax (Hs00180269_m1), GAPDH (Hs00266705_g1), MDM2 (Hs01066930_m1), and HMGB1 (Hs01923466_g1).

Techniques: Infection, Activation Assay

Journal: Scientific Reports

Article Title: Identification and functional interpretation of miRNAs affected by rare CNVs in CAKUT

doi: 10.1038/s41598-022-22749-1

Figure Lengend Snippet: The difference in the relative expression of target genes mRNA between MIR484 +/− KO and HEK293 WT. Relative mRNA levels were standardized against GAPDH endogenous control and presented as a scatter plot of 2 −dCt values with standard errors of the mean from three independent replicates. ( A ) Relative levels of MDM2 mRNA , Student's t-test, P = 0.005. ( B ) Relative levels of APAF1 mRNA, Student's t-test, P < 0.0001 ( C ) Relative levels of NOTCH3 mRNA, Student's t-test, P = 0.014. ( D ) Relative levels of FIS1 mRNA, Student's t-test, P = 0.081. ( E ) Relative levels of PKD1 mRNA, Student's t-test, P = 0.742. * denotes a significant difference at P < 0.05; ** denotes a significant difference at P < 0.01; ns denotes a nonsignificant difference in relative target gene mRNA levels.

Article Snippet: Expression levels of the selected hsa-miR-484 target genes were measured by quantitative Real-time PCR on Applied Biosystems Real-Time 7500 system (Applied Biosystems, Inc., Foster City, CA) using TaqMan ® gene expression assay: Hs00947377_m1 (for PKD1 (polycystin-1)), Hs00540450_s1 (for MDM2 (Mouse double minute 2 homolog)), Hs00211420_m1 (for FIS1 (Mitochondrial Fission 1 Protein)), Hs01128537_m1 (for NOTCH3 (Neurogenic locus notch homolog)), Hs00559441_m1 (for APAF1 (Apoptosis protease-activating factor-1)), Hs02786624_g1 (for GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)).

Techniques: Expressing, Control

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: A Representative western blots of p53 and LIN28B expression in A2780 and TOV-112D cells with LIN28B knockdown (sh LIN28B ) and overexpression (LIN28B) from three independent experiments. B Representative western blots of p53 and LIN28B expression in LOXI and CRMM2 cells with LIN28B knockdown (sh LIN28B ) from three independent experiments. C Correlation of LIN28B and p53 protein expression in ovarian cancer cell lines ( n = 29) based on Supplementary Fig. and our previous publication . D Representative immunohistochemistry staining image of p53 in LIN28B knockdown (sh LIN28B ) and overexpression (LIN28B) xenograft tumors. Bar, 100 μm. E Representative western blots of MDM2 and MDM4 expression in A2780 and TOV-112D cells with LIN28B knockdown (sh LIN28B ) and overexpression (LIN28B) from three independent experiments. F qPCR of TP53 mRNA level in LIN28B knockdown (sh LIN28B ) and overexpression (LIN28B) samples. Ctrl = control. Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant. G , qPCR of TP53 mRNA in LIN28B knockdown (sh LIN28B ) and overexpression (LIN28B) cells treated by actinomycin D (10 μg/ml). Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant.

Article Snippet: The primary antibodies are as follows: β-Actin (1:10,000; Sigma); α-Tubulin (1:5000; Sigma); LIN28B (1:1000; Cell Signaling Technology, #11965); TP53 (1:1000; Cell Signaling Technology, #2527); RPL26 (1:1000; Abcam, ab59567); cleaved caspase-3 (1:1000; Cell Signaling Technology, #9664); Nucleolin (1:1000; Cell Signaling Technology, #14574); MDM2 (1:1000; Cell Signaling Technology, #51541); MDM4 (1:1000; Sigma-Aldrich, 04-1556); and LaminB1 (1:1000; Abcam, ab16048).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Immunohistochemistry, Staining, Control

Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: A Relative mRNA level of LIN28B in control (DMSO) and p53–MDM2 inhibitor Nutlin3a, RG7388 (RG, Idasanutlin) treated cells by analyzing the GSE154065 (HCT116) and GSE104917 (NB1691 neuroblastoma) datasets. * p < 0.05, ** p < 0.01. B Relative cell viability as determined by CCK8 in ocular melanoma cells treated by various concentrations of Nutlin3a and MI773. C Correlation between relative cell viability and LIN28B protein level in ocular melanoma cells treated by Nutlin3a (22.2 μM). D Correlation between Nutlin3a IC50 and LIN28B protein level in ocular melanoma cells. E Correlation between relative cell viability and LIN28B protein level in ocular melanoma cells treated by MI773 (22.2 μM). F Correlation between MI773 IC 50 and LIN28B protein level in ocular melanoma cells treated. G Relative cell viability as determined by CCK8 in ocular melanoma cells treated by various concentrations of Siremadin and Idasanutlin (RG7388). Representative data of three independent experiments (mean ± s.e.m.). H Relative cell viability of control (shCtrl) and knockdown (shLIN28B) LOXI cells treated by various concentrations of Nutlin3a. The IC 50 of each group was shown. Representative data of three independent experiments (mean ± s.e.m.). I Schematic diagram of the working model.

Article Snippet: The primary antibodies are as follows: β-Actin (1:10,000; Sigma); α-Tubulin (1:5000; Sigma); LIN28B (1:1000; Cell Signaling Technology, #11965); TP53 (1:1000; Cell Signaling Technology, #2527); RPL26 (1:1000; Abcam, ab59567); cleaved caspase-3 (1:1000; Cell Signaling Technology, #9664); Nucleolin (1:1000; Cell Signaling Technology, #14574); MDM2 (1:1000; Cell Signaling Technology, #51541); MDM4 (1:1000; Sigma-Aldrich, 04-1556); and LaminB1 (1:1000; Abcam, ab16048).

Techniques: Control, Knockdown

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