Journal: Journal of Functional Foods

Article Title: Krill oil attenuates diabetes-associated cognitive dysfunction by inhibiting microglial polarization-induced neuron injury

doi: 10.1016/j.jff.2024.106064

Figure Lengend Snippet: Fig. 5. KO had no protective effect on AGEs-activated neuron apoptosis. (A) Diagram for culture and treatment of N2a cells; (B) TUNEL staining with (C) percentage of positive cells quantified; (D) protein levels of BAX, BCL2 and cleaved Caspase3. The data were summarized as means ± SD (n = 3). *P < 0.05 vs. Ctrl; #P < 0.05 vs. AGEs + Veh. Abbreviations: AGEs, advanced glycation end products; Veh, vehicle (glycerol). Other abbreviations are the same as in Fig. 4.

Article Snippet: Mouse neuron N2a cells were obtained from The American Type Culture Collection (ATCC, CCL131, Manassas, Virginia, USA).

Techniques: TUNEL Assay, Staining

Journal: The CRISPR Journal

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1089/crispr.2019.0062

Figure Lengend Snippet: Cloning duo-guides into AAV-v1 vector by exploiting built-in cloning site (originally designed for one single guide RNA [sgRNA]). Gene editing (amino acid [AA] changes at Psen1 gene) in N2A cell line. (A) Top: Schematic AAV-v1 construct: blue dotted line zooming out partial sequences of U6 and sgRNA cloning site. Middle: Type IIS RE (SapI) digest creates two unique 5′ overhangs (GGT vs. GTT). Bottom: Showing gene synthesized plasmid carrying duo-sgRNA cassette sequence (Spacer-W + tracrRNA + U6 + spacer-X) with complementary overhangs, flanked by uniquely positioned SapI sites, which upon SapI digestion renders two complementary 5′ overhangs (ACC vs. AAC; red dotted line) for cloning. (B) Schematic of the final construct AAV-v2. (C) Top: CRISPR guides (W and X) target sites (purple scissors) on Psen1 exon 10 region. Bottom: AA replacement donor (R) integrated at the genomic target after rAAV-v2 transduction. (D) Psen1 exon 10 sequence shown (WT vs. R: 3/5 AA changes—three amino acid codons yellow highlighted). Red arrow: SpCas9 guides (W and X) cutting sites; chromatograms showing WT, mutant (R) and hemizygous (R + indel); green dotted rectangle encompassing the 3/5 AA changes within a 15 nt range.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo; cat. # SL511; GeneCopoeia) was cultured in medium, comprising 44% Dulbecco's modified Eagle's Medium (cat. # 30-2002; ATCC), 50% Opti-MEM (cat. # 51985-034; Life Technologies) with 5% fetal bovine serum (cat. # 100-500; Gemini), and 1% penicillin-streptomycin (cat. # P4333; Millipore Sigma) in a 37°C humid incubator with 5% CO 2 .

Techniques: Cloning, Plasmid Preparation, Construct, Synthesized, Sequencing, CRISPR, Transduction, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity

doi: 10.3389/fphar.2024.1459909

Figure Lengend Snippet: Oleuropein reduces mutant huntingtin aggregates. (A) Images showing ST Hdh Q7/Q7 striatal cells co-transfected with the HttQP72-GFP and mCherry plasmids after oleuropein treatment. Cells without treatment were used as the control. (B) Quantification analysis of the percentage of cells containing the aggregates in ST Hdh Q7/Q7 striatal cells transfected with the indicated plasmids in the absence or presence of oleuropein treatment at the indicated concentration for 24 h. (C) Immunoblot detection of the aggregated and soluble forms of HttQP72-GFP in N2a cells transfected with the indicated plasmid incubated in the absence or presence of oleuropein at the indicated concentration for 24 h. Tubulin protein was used as an internal control. (D) Quantification analyses of the normalized HttQP72-GFP aggregates. Tubulin was used as an internal control for normalization in (C) . Data from three independent experiments are presented as mean normalized units ± SEM. Data showing significant differences are labeled as follow: p < 0.05 with one asterisk ( * ), and p < 0.01 with two asterisks ( ** ). N.S. , no significance.

Article Snippet: Mouse neuroblastoma Neuro-2a (N2a) cell line was purchased from Bioresource Collection and Research Center, Taiwan (RRID: CVCL_0470, BCRC, Cat#60026).

Techniques: Mutagenesis, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Incubation, Labeling

Journal: Frontiers in Pharmacology

Article Title: Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity

doi: 10.3389/fphar.2024.1459909

Figure Lengend Snippet: Oleuropein decreases the aggregated and soluble forms of truncated mHtt in N2a cells. (A) Immunoblot detection of the aggregated and soluble forms of mutant Htt (HttQP72-GFP) in N2a cells transfected with the HttQP72-GFP plasmid in the absence or presence of oleuropein treatment at the indicated concentration for 24 or 48 h. Cells were treated with 100 μg/ml cycloheximide (CHX) to inhibit protein synthesis. Actin was used as an internal control. (B) Quantification analyses of the aggregated form of HttQP72-GFP protein. (C) Quantification analyses of the soluble form of HttQP72-GFP protein. Actin was used for normalization. Data from three independent experiments are presented as mean normalized units ± SEM. Data showing significant differences are labeled as follows: p < 0.05 with one asterisk ( * ) and p < 0.01 with two asterisks ( ** ).

Article Snippet: Mouse neuroblastoma Neuro-2a (N2a) cell line was purchased from Bioresource Collection and Research Center, Taiwan (RRID: CVCL_0470, BCRC, Cat#60026).

Techniques: Western Blot, Mutagenesis, Transfection, Plasmid Preparation, Concentration Assay, Control, Labeling

Journal: Frontiers in Pharmacology

Article Title: Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity

doi: 10.3389/fphar.2024.1459909

Figure Lengend Snippet: Oleuropein does not affect autophagic flux in HD model cells. (A) Immunoblotting analysis of the ST Hdh Q7/Q7 and ST Hdh Q111/Q111 striatal cells treated with 5 μg/ml oleuropein or 200 nM Bafilomycin A1 or both. Immunoblot detection of Htt, LC3, and p62 proteins in ST Hdh Q7/Q7 and ST Hdh Q111/Q111 striatal cells. Actin was used as an internal control. Quantitation analyses of (B) LC3-II or (C) p62 protein normalized to actin. (D) Immunoblotting analysis of LC3 and p62 in N2a cells expressing truncated wild-type or mutant Htt after treatment with 5 μg/ml oleuropein or 200 nM Bafilomycin A1 or both. Actin protein was used as an internal control. Quantitation analysis of (E) LC3II or (F) p62 protein normalized to actin. Data from three independent experiments are presented as mean normalized units ± SEM. Data showing significant differences are labeled as follows: p < 0.05 with one asterisk ( * ), p < 0.005 with two asterisks ( ** ), and p < 0.001 with three asterisks ( *** ). N.S. , not significant.

Article Snippet: Mouse neuroblastoma Neuro-2a (N2a) cell line was purchased from Bioresource Collection and Research Center, Taiwan (RRID: CVCL_0470, BCRC, Cat#60026).

Techniques: Western Blot, Control, Quantitation Assay, Expressing, Mutagenesis, Labeling

Journal: Frontiers in Pharmacology

Article Title: Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity

doi: 10.3389/fphar.2024.1459909

Figure Lengend Snippet: Oleuropein increases the proteasome activity in N2a cells expressing the truncated HttQP72-GFP mHtt protein. Oleuropein increases the proteasome activity in N2a cells expressing the truncated HttQP72-GFP mHtt protein. The chymotrypsin-like activity (A) , caspase-like activity (B) , and trypsin-like activity (C) were examined in N2a cells expressing wild-type or the truncated mHtt in the absence or presence of 5 μg/ml oleuropein treatment at the indicated concentration for 24 h. Data from three independent experiments are presented as mean normalized units ± SEM. Data showing a significant difference ( p < 0.05) is labeled with one asterisk ( * ). N.S. , not significant.

Article Snippet: Mouse neuroblastoma Neuro-2a (N2a) cell line was purchased from Bioresource Collection and Research Center, Taiwan (RRID: CVCL_0470, BCRC, Cat#60026).

Techniques: Activity Assay, Expressing, Concentration Assay, Labeling

Journal: Frontiers in Pharmacology

Article Title: Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity

doi: 10.3389/fphar.2024.1459909

Figure Lengend Snippet: Effects of oleuropein on the proteasome-associated proteins in N2a cells expressing the truncated HttQP72-GFP mHtt protein. (A) Immunoblot detection of Htt, β1, β2, β5, PA28γ, phospho-p38, and p38 in N2a cells expressing truncated wild-type or mutant Htt in the absence or presence of oleuropein treatment at the indicated concentration for 24 h. Actin was used as an internal control. (B–H) Quantification analyses of protein levels with the indicated protein normalized to the actin protein. A paired Student’s t-test was used for statistical analysis. Data showing significant differences compared to the control are labeled as follows: p < 0.05 with one asterisk ( * ), and p < 0.01 with two asterisks ( ** ). N.S. , no significance.

Article Snippet: Mouse neuroblastoma Neuro-2a (N2a) cell line was purchased from Bioresource Collection and Research Center, Taiwan (RRID: CVCL_0470, BCRC, Cat#60026).

Techniques: Expressing, Western Blot, Mutagenesis, Concentration Assay, Control, Labeling

Journal: Endocrinology

Article Title: Glucocorticoid Receptor–Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses

doi: 10.1210/en.2018-00819

Figure Lengend Snippet: ChIP-nexus identifies GR and MR binding sites in N2A cells. (a and b) ChIP-qPCR showing specificity of (a) GFP and (b) RFP antibodies. N2A cells transiently transfected with EGFP-GR show the ChIP-qPCR signal detected by the GFP antibody with minimal cross-reactivity to the RFP antibody. Transfection with mCherry-MR shows the ChIP-qPCR signal detected by the RFP antibody with minimal cross-reactivity to the GFP antibody. EGFP-GR and mCherry-MR were cotransfected with shRNA to NR3C1 -3′UTR to minimize possible endogenous GR effects, treated with 100 nM CORT for 20 min, and primers were located at the Sgk1 gene. Data are represented as means relative to percentage input ± SEM (n = 3; one-way ANOVA with a Tukey test). (c) Graph shows the distribution of MACS2-identified GR and MR binding sites within known genes, 5 kb upstream, 5 kb downstream, and intergenic regions for each treatment group: (i) vehicle for 20 min, (ii) 100 nM CORT for 20 min, and (iii) 100 nM CORT for 20 min, washout, and further incubation for 40 min. (d) Graph shows the number of genes with single or multiple associated binding sites for GR/MR with treatment (ii). (e and f) Area-proportional Venn diagrams show the proportions of GR and MR MACS2 binding sites that directly overlap by at least 1 bp between treatments (i), (ii), and (iii). (g) University of California Santa Cruz Genome Browser image at the Fkbp5 gene shows comparison of mapped MR and GR ChIP-nexus data for each treatment group. *** P < 0.001; **** P < 0.0001. Ab, antibody; veh, vehicle.

Article Snippet: Authenticated Neuro 2A (N2A) mouse neuroblast cells from the European Collection of Authenticated Cell Cultures [RRID: CVCL_0470 ( )] were purchased (January 2015) from Sigma-Aldrich (St. Louis, MO) and cultured up to passage 20, during which they showed consistent characteristic neuronal morphology and, upon differentiation stimulus , were capable of producing neurite projections (last tested September 2018).

Techniques: Binding Assay, ChIP-qPCR, Transfection, shRNA, Incubation, Comparison

Journal: Endocrinology

Article Title: Glucocorticoid Receptor–Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses

doi: 10.1210/en.2018-00819

Figure Lengend Snippet: MR and GR binding sites are highly similar. (a) Area-proportional Venn diagram shows MACS2 binding sites for MR that directly overlap with GR binding sites by at least 1 bp after 20 min of CORT (treatment ii). (b and c) University of California Santa Cruz Genome Browser images of mapped ChIP-nexus data and MACS2 peaks for MR/GR in mCherry-MR– and EGFP-GR–transfected N2A cells after 20 min of CORT (treatment ii) at the (b) Syt2 gene and (c) Ddc gene. (d) Diagram shows how λ -exonuclease digestion of ChIP fragments defines the binding regions. (e) Distribution of coverage for the MACE-predicted binding regions for GR and MR after CORT for 20 min (treatment ii) shows highly similar binding locations.

Article Snippet: Authenticated Neuro 2A (N2A) mouse neuroblast cells from the European Collection of Authenticated Cell Cultures [RRID: CVCL_0470 ( )] were purchased (January 2015) from Sigma-Aldrich (St. Louis, MO) and cultured up to passage 20, during which they showed consistent characteristic neuronal morphology and, upon differentiation stimulus , were capable of producing neurite projections (last tested September 2018).

Techniques: Binding Assay, Transfection

Journal: Endocrinology

Article Title: Glucocorticoid Receptor–Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses

doi: 10.1210/en.2018-00819

Figure Lengend Snippet: Differential effects of GR and MR on gene expression. (a–d) Graphs show (a) Syt2 mRNA, (b) Sgk1 mRNA, (c) Dusp4 mRNA, and (d) Ddc nascent RNA changes in N2A cells with expressed wild-type MR and GR individually or combined, and MR-XDBD/GR-XDBD mutants individually or in combination with GR/MR. RT-qPCR data from N2A cells treated with 100 nM CORT for 2 h after transient transfection for 24 h with MR/GR/MR+GR expression plasmids and cotransfection of NR3C1 -3′UTR shRNA to minimize endogenous GR effects. Data are represented as mean fold changes relative to EGFP-transfected controls ± SEM (n ≥ 3). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Authenticated Neuro 2A (N2A) mouse neuroblast cells from the European Collection of Authenticated Cell Cultures [RRID: CVCL_0470 ( )] were purchased (January 2015) from Sigma-Aldrich (St. Louis, MO) and cultured up to passage 20, during which they showed consistent characteristic neuronal morphology and, upon differentiation stimulus , were capable of producing neurite projections (last tested September 2018).

Techniques: Gene Expression, Quantitative RT-PCR, Transfection, Expressing, Cotransfection, shRNA

Journal: Endocrinology

Article Title: Glucocorticoid Receptor–Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses

doi: 10.1210/en.2018-00819

Figure Lengend Snippet: MR is tethered to genomic DNA by GR. (a–d) ChIP-qPCR shows mCherry-MR-XDBD binding to chromatin without/with cotransfected wild-type GR or MR at the (a) Syt2 , (b) Sgk1 , (c) Dusp4 , and (d) Ddc binding sites. (e–h) ChIP-qPCR shows EGFP-GR-XDBD binding to chromatin with/without cotransfected wild-type MR or GR at the (e) Syt2 , (f) Sgk1 , (g) Dusp4 , and (h) Ddc binding sites. N2A cells were treated with 100 nM CORT for 20 min (a–h). (i and j) ChIP-qPCR shows mCherry-MR-XDBD binding to chromatin at the (i) Syt2 and (j) Ddc binding sites with treatments: vehicle for 20 min, 100 nM CORT for 20 min, or 100 nM CORT for 20 min, washout, and further incubation for 40 min. Data are taken from representative ChIP experiments (n = 3) expressed as a percentage of input. N2A cells were transiently transfected with mCherry-MR(wild-type)/mCherry-MR-XDBD/MR and EGFP-GR/EGFP-GR-XDBD/GR or an EGFP control, with shRNA to endogenous NR3C1 -3′UTR. Ab, antibody; veh, vehicle; wt, wild-type.

Article Snippet: Authenticated Neuro 2A (N2A) mouse neuroblast cells from the European Collection of Authenticated Cell Cultures [RRID: CVCL_0470 ( )] were purchased (January 2015) from Sigma-Aldrich (St. Louis, MO) and cultured up to passage 20, during which they showed consistent characteristic neuronal morphology and, upon differentiation stimulus , were capable of producing neurite projections (last tested September 2018).

Techniques: ChIP-qPCR, Binding Assay, Incubation, Transfection, Control, shRNA

Journal: Endocrinology

Article Title: Glucocorticoid Receptor–Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses

doi: 10.1210/en.2018-00819

Figure Lengend Snippet: Effects of MR-A640T mutation on gene expression. (a and b) Graphs show (a) Syt2 mRNA and (b) Ddc nascent RNA changes in N2A cells with expressed wild-type GR and MR/MR-XDBD/MR-A640T/MR-XDBD-A640T individually or combined. RT-qPCR data are shown from N2A cells treated with 100 nM CORT for 2 h after transient transfection for 24 h with MR/GR/MR+GR expression plasmids and cotransfection of NR3C1 -3′UTR shRNA to minimize endogenous GR effects. Data are represented as mean fold changes relative to EGFP-transfected controls ± SEM (n ≥ 3). * P < 0.05; *** P < 0.001.

Article Snippet: Authenticated Neuro 2A (N2A) mouse neuroblast cells from the European Collection of Authenticated Cell Cultures [RRID: CVCL_0470 ( )] were purchased (January 2015) from Sigma-Aldrich (St. Louis, MO) and cultured up to passage 20, during which they showed consistent characteristic neuronal morphology and, upon differentiation stimulus , were capable of producing neurite projections (last tested September 2018).

Techniques: Mutagenesis, Gene Expression, Quantitative RT-PCR, Transfection, Expressing, Cotransfection, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

doi: 10.1074/jbc.RA118.006583

Figure Lengend Snippet: Acute ethanol treatment reduces the total level of β-arrestin2 protein in N2A-5HT1AR cells and rat PFC. A, representative Western blots and quantification of β-arrestin2 (β-arr2) protein levels in N2A-5HT1AR cells. Cells were acutely treated with ethanol (15–75 mm) or vehicle (Veh; media) for 18 h. The 30 and 75 mm ethanol treatment significantly reduced β-arrestin2 protein levels in N2A-5HT1AR cells (n = 6, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). B, representative Western blots and quantification of β-arrestin 1/2 protein levels in rat PFC. Rats were acutely exposed to ethanol vapor (EtOH) or control (Air) for 12 h followed by immediate sacrifice. Ethanol exposure significantly reduced β-arrestin2 protein level in rat PFC (n = 6, *, p < 0.05 versus air, unpaired Student's t test).

Article Snippet: Immunoprecipitates and N2A cell lysates were immunoblotted ( IB ) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide.

Techniques: Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

doi: 10.1074/jbc.RA118.006583

Figure Lengend Snippet: Acute ethanol exposure increases ubiquitination of β-arrestin2 in N2A-5HT1AR cells and rat PFC. A, representative raw and binary fluorescent images of β-arrestin2 (green) and ubiquitin (red) and co-localization (Co-loc) in N2A-5HT1AR cells treated with ethanol (15–75 mm) or vehicle (Veh) (media) for 18 h. B, ethanol exposure dose-dependently increased β-arrestin2 co-localization with ubiquitin (n = 29–30 cells/group, six replicates, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). C, validation of the rabbit anti-β-arrestin2 antibody (LSBio; LS-B15546) was performed using a custom-made blocking peptide (GenScript; sequence DDIVFEDFARLRLK). Immunoprecipitation (IP) was performed using lysates from drug-naïve N2A-5HT1AR cells and rat PFC tissue in the presence and absence of the corresponding β-arrestin2 (β-arr)-blocking peptide (10 μg). Immunoprecipitates and N2A cell lysates were immunoblotted (IB) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide. D, representative blots for immunoprecipitated β-arrestin2, ubiquitin, and MDM2 from N2A-5HT1AR cell lysates. Cells were treated with ethanol (30 mm, 18 h) or vehicle (Veh) (media), and cell lysates were immunoprecipitated overnight with rabbit anti-β-arrestin2 antibody. Immunoprecipitates and cell lysates were immunoblotted with mouse anti-ubiquitin, mouse anti-MDM2, rabbit anti-NEDD4, and mouse anti-β-arrestin2 antibodies. E–H, quantification of β-arrestin2 mono- and poly-ubiquitination, MDM2, and NEDD4 (n = 5, **, p < 0.01 versus vehicle, unpaired Student's t test). I, representative blots for immunoprecipitated β-arrestin2, ubiquitin, and MDM2 in rat PFC lysates. Samples from ethanol (EtOH) and control (Air) exposed rats were immunoprecipitated with rabbit anti-β-arrestin2 overnight. Immunoprecipitates and tissue lysates were immunoblotted with the antibodies described above. J–M, quantification of immunoprecipitated β-arrestin2, ubiquitin, MDM2, and NEDD4 (n = 5, *, p < 0.05 versus vehicle, unpaired Student's t test).

Article Snippet: Immunoprecipitates and N2A cell lysates were immunoblotted ( IB ) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide.

Techniques: Ubiquitin Proteomics, Biomarker Discovery, Blocking Assay, Sequencing, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

doi: 10.1074/jbc.RA118.006583

Figure Lengend Snippet: Acute ethanol exposure reduces β-arrestin2 protein expression in an MDM2- and proteasome-dependent manner in N2A-5HT1AR cells. A, verification of MDM2 protein knockdown via siRNA. N2A-5HT1AR cells were transfected with MDM2 siRNA or a corresponding scramble siRNA. MDM2 protein levels were measured 48 h after transfection by Western blotting. MDM2 siRNA treatment significantly reduced the endogenous MDM2 protein level when compared with scrambled siRNA-treated control cells (n = 5, **, p < 0.01 versus scramble, unpaired Student's t test). B, representative Western blotting of β-arrestin2 (β-arr2) protein level in scramble and MDM2 siRNA-treated cells. N2A-5HT1AR cells were transfected with MDM2 siRNA or a corresponding scramble siRNA and then treated with ethanol (30–75 mm) or vehicle (Veh) (media) for 18 h. Cell lysates were immunoblotted with rabbit anti-β-arrestin2 and goat anti-β-actin antibodies. C, quantification of β-arrestin2 levels in scramble and MDM2 siRNA-treated cells. MDM2 knockdown prevented ethanol-induced degradation of β-arrestin2 (n = 6, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test). D, schematic illustration of spatial overlap analysis for quantitation of ubiquitin, β-arrestin2, and P19S co-localization. Co-localization between β-arrestin2 (green) and ubiquitin (red) was first determined as yellow overlapped pixels (left, middle). These co-localized pixels (yellow; left, bottom) were then isolated, and triple co-localization with P19S (blue; right, top) was determined as white overlapped pixels (right, bottom). Data were reported as a percentage of co-localization by overlapping pixel counts, y/x + y + z (ubiquitin/β-arrestin2 co-localization with respect to P19S). E, representative confocal images of ubiquitin (red), β-arrestin2 (green), and P19S (blue) in N2A-5HT1AR cells after acute treatment with ethanol (15–75 mm,18 h) or vehicle (media). Scale bars, 10 μm. F, quantification of ubiquitin, β-arrestin2, and P19S co-localization. The co-localization of the ubiquitin, β-arrestin2, and P19S significantly increased after ethanol treatment (30–75 mm) (n = 25–28 cells/group, five replicates, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). G, representative Western blotting of β-arrestin2 levels in N2A-5HT1AR incubated with or without MG132 (5 μm) during treatment with ethanol (30–75 mm, 18 h) or vehicle (media). H, quantification of β-arrestin2 protein levels. Treatment with MG132 prevented acute ethanol-induced (30 and 75 mm) reduction in β-arrestin2 protein levels in N2A-5HT1AR cells (n = 5, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test). I, representative confocal images of LAMP1 (red) and β-arrestin2 (green) in N2A-5HT1AR cells after acute treatment with ethanol (30–75 mm, 18 h) or vehicle (media). Scale bars, 10 μm. J, quantification of β-arrestin2 and LAMP1 co-localization. β-Arrestin2 co-localization with LAMP1 significantly increased after a high-dose ethanol treatment (75 mm) compared with vehicle treatment (n = 35 cells/group, five replicates, *, p < 0.05 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test).

Article Snippet: Immunoprecipitates and N2A cell lysates were immunoblotted ( IB ) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide.

Techniques: Expressing, Knockdown, Transfection, Western Blot, Control, Quantitation Assay, Ubiquitin Proteomics, Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

doi: 10.1074/jbc.RA118.006583

Figure Lengend Snippet: Acute ethanol exposure inhibits agonist-induced 5-HT1AR internalization. N2A-5HT1AR cells were acutely treated with ethanol (15–75 mm, 18 h) or vehicle (Veh) (media) followed by labeling the surface 5-HT1ARs with mouse anti-HA antibody. Then cells were warmed to 37 °C and treated with 8-OH-DPAT (1 μm) for the indicated time points to induce internalization. At the end of each time point, the remaining primary antibody-bound surface 5-HT1ARs that did not internalize were labeled with goat anti-mouse Alexa 647 (red). Internalized intracellular 5-HT1ARs were identified by labeling with goat anti-mouse Alexa 405 (blue, pseudo-colored to green). A, representative confocal images of surface and internalized 5-HT1ARs from vehicle- and ethanol-treated cells (15–75 mm). Scale bar, 10 μm. B, quantification of 8-OH-DPAT–stimulated 5-HT1AR internalization. Internalized 5-HT1ARs were calculated as percent of their own total surface 5-HT1AR intensity. Receptor internalization occurred in a time-dependent manner in both vehicle- and ethanol-treated cells (n = 47–50 cells/group, five replicates, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test). Ethanol treatment (30–75 mm) significantly inhibited 8-OH-DPAT–stimulated 5-HT1AR internalization compared with the vehicle treatment. Data are normalized and expressed relative to vehicle. C, MDM2 siRNA knockdown blocked the inhibition of 5-HT1AR internalization after ethanol (30–75 mm, 18 h) or vehicle (media) treatment (n = 35–38 cells/group, six replicates, **, p < 0.01 versus scramble, a two-way ANOVA followed by Bonferroni post hoc test).

Article Snippet: Immunoprecipitates and N2A cell lysates were immunoblotted ( IB ) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide.

Techniques: Labeling, Knockdown, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

doi: 10.1074/jbc.RA118.006583

Figure Lengend Snippet: Acute ethanol exposure reduces and delays recruitment of β-arrestin2 to the plasma membrane. N2A-5HT1AR cells were treated with ethanol (15–30 mm, 18 h) or vehicle (Veh) (media) followed by stimulation with 8-OH-DPAT (1 μm for 5, 15, or 30 min). Surface 5-HT1ARs and cytosolic β-arrestin2 were fluorescently labeled as described under “Experimental procedures.” A, schematic illustration of manual segmentation for quantitation of β-arrestin2 (β-arr2) translocation toward and away from the membrane. The outer boundary of each cell was manually outlined based on the localization of surface 5-HT1AR staining (left, red). An inner boundary was defined as a standardized 2-μm distance from the outer boundary. The outer sector was determined as the area between the outer and inner boundary, and the inner sector was determined as the remaining cellular area toward the cell center starting at the indicated inner boundary (right). β-Arrestin2 translocation from the membrane to the cytoplasm was calculated as the ratio of the outer to the inner sector intensity. B, representative confocal images of β-arrestin2 localization from ethanol (15–30 mm) and vehicle (Veh) (media)-treated cells. Scale bars, 10 μm. C, quantification of β-arrestin2 membrane localization. The level of β-arrestin2 membrane localization following 30 min of 8-OH-DPAT stimulation was reduced in cells treated with 30 mm ethanol compared with vehicle treatment (n = 28–30 cells/group, five replicates, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). D, ethanol treatment (30 mm) delayed β-arrestin2 membrane translocation. 8-OH-DPAT stimulation to control cells significantly increased the redistribution of β-arrestin2 into the outer segment as early as 5 min; however, 8-OH-DPAT stimulation to ethanol-treated cells (30 mm, 18 h) did not increase β-arrestin2 redistribution into the outer segment until 15 min (n = 28–30 cells/group, five replicates, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test).

Article Snippet: Immunoprecipitates and N2A cell lysates were immunoblotted ( IB ) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide.

Techniques: Clinical Proteomics, Membrane, Labeling, Quantitation Assay, Translocation Assay, Staining, Control