n2a parental cell line  (ATCC)

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: Generation of N2A-5-HT 1A R stable cell line Parental N2A cells (ATCC) were grown in Opti-MEM media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

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: Generation of N2A-5-HT 1A R stable cell line Parental N2A cells (ATCC) were grown in Opti-MEM media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

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: Generation of N2A-5-HT 1A R stable cell line Parental N2A cells (ATCC) were grown in Opti-MEM media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

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: Generation of N2A-5-HT 1A R stable cell line Parental N2A cells (ATCC) were grown in Opti-MEM media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

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: Generation of N2A-5-HT 1A R stable cell line Parental N2A cells (ATCC) were grown in Opti-MEM media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

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