Journal: Nature Communications

Article Title: Non-canonical interplay between glutamatergic NMDA and dopamine receptors shapes synaptogenesis

doi: 10.1038/s41467-023-44301-z

Figure Lengend Snippet:

Article Snippet: rabbit anti-D2R , 55084-1-AP , Proteintech , 2 μg.

Techniques:

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 1. DMCs identified in the DRD2 promoter region of breast cancer tissues (C1-C7) to the paired adjacent tissues (N1-N7). (A) A heatmap of the 8 DMCs in the breast cancer tissues. (B) The mean methylation level, differences, and significance level of these DMCs. (C) The methylation level of these DMCs in cancer tissues and their paired adjacent tissues (negative control).

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Methylation, Negative Control

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 3. Construction of DRD2 overexpression and downregulation models. (A) Representative immunoblot of DRD2 expression of the original MCF-7 cells (Con), MCF-7 with blank vector (NC), and MCF-7 with DRD2 overexpression (OE). (B) The quantification of DRD2 in MCF-7 cells by western blot. (C) Representative immunoblot of DRD2 expression of the original MB231 cells (Con), MB231 with non-target siRNA (shNC), and MB231 with three different shRNAs (shRNA1, shRNA2, shRNA3). (D) The quantification of DRD2 in MB231 cells by western blot. * vs Hs 578Bst/con, P<0.05; # vs NC/shNC, P < 0.05.

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 2. The expression levels of DRD2 in breast cancer cells. (A) Representative immunoblot of DRD2 expression of the breast cancer cells (MCF-7, SK-BR3, and MB231) and normal breast cells (Hs 578Bst). (B-C) The relationship between DRD2 and β-actin was detected by PCR, and the relationship between DRD2 and α-tublin was detected by western blot. * vs Hs 578Bst, P<0.05.

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Expressing, Western Blot

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 4. DRD2 promotes the proliferation and migration of breast cancer cells. (A) Proliferation of MB231 treated with different concentrations of 5-AzadC. (B) Proliferation of original MCF-7 cells (Con), MCF-7 with blank vector (NC), and MCF-7 with DRD2 overexpression (OE). (C) Proliferation of original MB231 cells (Con), MB231 with non-target siRNA (shNC), MB231 with DRD2 shRNA (shRNA), and MB231 with DRD2 shRNA plus 5-AzadC (shRNA + 5-AzadC). (D) Statistics for migration of Con, NC, and OE cells. (E) Representative cell images for the migration ability of Con, NC, and OE cells. (F) Statistics for migration of Con, shNC, shRNA, and shRNA + 5-AzadC cells. (G) Representative cell images for the migration ability of Con, shNC, shRNA, and shRNA + 5-AzadC cells. * vs con, P < 0.05; # vs NC/ shNC, P < 0.05; $ vs shRNA, P < 0.05.

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Migration, Plasmid Preparation, Over Expression, shRNA

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 5. DRD2 promotes cancer through ERK signaling pathway. (A) Western blot for DRD2, ERK and p-ERK expression in primary MCF-7 cells (Con), blank vector (NC) and DRD2 overexpressing (OE). (B-E) Quantitative analysis of FLNA, ERK, p-ERK and DRD2 expression in different MCF-7 cells. (F) Western blot of DRD2 expression in primary MB231 cells (Con), MB231 and non-target siRNA (shNC), MB231 and DRD2 shRNA (shRNA), MB231 and DRD2 shRNA + 5-AzadC (shRNA + 5- AzadC) . (G-J) Quantitative analysis of FLNA, ERK, p-ERK and DRD2 expression in different MB231 cells.

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Western Blot, Expressing, Plasmid Preparation, shRNA

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 6. The expression of DRD2 regulated tumor growth in vivo. (A, D) Subcutaneous xenograft model images of blank vector (NC) and DRD2-overexpressing MCF-7 cells (OE) in nude mice and their tumors. (B) Tumor growth curves of NC group and OE group. (C) Tumor weights in NC group and OE group. (E) Expression of Ki67 and CD31 in NC and OE tissues.

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Expressing, In Vivo, Plasmid Preparation

Journal: Cancer genetics

Article Title: Hypomethylation of DRD2 promotes breast cancer through the FLNA-ERK pathway.

doi: 10.1016/j.cancergen.2023.09.001

Figure Lengend Snippet: Fig. 7. Detection of protein levels in subcutaneous tumor tissue. (A) Western blot analysis of FLNA, DRD2, ERK and p-ERK expression in MCF-7-NC and MCF-7-OE tissues. (B) Quantitative analysis of FLNA, ERK, p-ERK and DRD2 expression in MCF-7-NC and MCF-7-OE tissues. (C) Representative immunoblots of FLNA, DRD2, ERK and p-ERK expression in MB231-shNC, MB231-shRNA and MB231-shRNA+5-AzadC tissues. (D) Quantitative analysis of FLNA, ERK, p-ERK, DRD2 expression in MB231-shNC, MB231-shRNA, MB231-shRNA+5-AzadC tissues.

Article Snippet: Antibodies against DRD2 (SC-5303, Santa, USA), ERK (4695S, CST, USA), p-ERK (4370S, CST, USA), and α-tublin (66,031–1-ig, proteintech, USA) were used for Western blot analysis.

Techniques: Western Blot, Expressing, shRNA

Journal: ACS Chemical Neuroscience

Article Title: Developing Photoaffinity Probes for Dopamine Receptor D 2 to Determine Targets of Parkinson’s Disease Drugs

doi: 10.1021/acschemneuro.2c00544

Figure Lengend Snippet: Intracellular calcium flux assay. HEK293T cell line stably expressing constructs for human DRD2 and a chimeric G-protein is loaded with calcium-sensing dye, Fura-4. After dosing the probe, confocal microscopy is used to determine the calcium flux in the cell by change in dye fluorescence. EC 50 curves determined with GraphPad software using a Hill slope of 1.0.

Article Snippet: Following blocking, the membrane was incubated with either 1 μg/mL Anti-Strep Tag II rabbit polyclonal antibody (Abcam, ab76949) or 2 μg/mL Anti-DRD2 rabbit polyclonal antibody (AbClonal, A12930) in TBST with 5% BSA overnight at 4 °C.

Techniques: Calcium Flux Assay, Stable Transfection, Expressing, Construct, Confocal Microscopy, Fluorescence, Software

Journal: ACS Chemical Neuroscience

Article Title: Developing Photoaffinity Probes for Dopamine Receptor D 2 to Determine Targets of Parkinson’s Disease Drugs

doi: 10.1021/acschemneuro.2c00544

Figure Lengend Snippet: β-Arrestin recruitment analysis. (a) PRESTO-TANGO assay schematic: a ligand binds a chimeric DRD2 receptor, which then recruits β-arrestin fused with a TEV protease. The protease cuts a site between the receptor and a fused transcription factor, which then transits to the nuclease to initiate transcription of a luciferase gene. The luciferase activity is subsequently quantified. (b) Agonism of β-arrestin recruitment is quantified in EC 50 curves via the detection of luciferase activity, using a Hill slope of 1.0.

Article Snippet: Following blocking, the membrane was incubated with either 1 μg/mL Anti-Strep Tag II rabbit polyclonal antibody (Abcam, ab76949) or 2 μg/mL Anti-DRD2 rabbit polyclonal antibody (AbClonal, A12930) in TBST with 5% BSA overnight at 4 °C.

Techniques: Luciferase, Activity Assay

Journal: ACS Chemical Neuroscience

Article Title: Developing Photoaffinity Probes for Dopamine Receptor D 2 to Determine Targets of Parkinson’s Disease Drugs

doi: 10.1021/acschemneuro.2c00544

Figure Lengend Snippet: Photo-cross-linking of dye-clicked probe: confocal microscopy. (a) Schematic of the methodology used in the labeling process. Cells stably expressing DRD2 fused to an N-terminal Strep Tag II are treated with DRD2-targeting probes 5 or 7 at 5 μM, photo-cross-linked, and excess probe is washed out. An Alexa Fluor 555 azide is then clicked to the probe, washed out, and cells are treated with an anti-Strep-Tag II antibody and fluorescent secondary to visualize DRD2. Nuclei were stained with DAPI. (b) Confocal microscopy results of labeled cells. All probes show some degree of labeling. However, probes 5 and 7 show a notable increase in the labeling density. Images taken with 40× magnification, scale bar: 10 μm.

Article Snippet: Following blocking, the membrane was incubated with either 1 μg/mL Anti-Strep Tag II rabbit polyclonal antibody (Abcam, ab76949) or 2 μg/mL Anti-DRD2 rabbit polyclonal antibody (AbClonal, A12930) in TBST with 5% BSA overnight at 4 °C.

Techniques: Confocal Microscopy, Labeling, Stable Transfection, Expressing, Strep-tag, Staining

Journal: ACS Chemical Neuroscience

Article Title: Developing Photoaffinity Probes for Dopamine Receptor D 2 to Determine Targets of Parkinson’s Disease Drugs

doi: 10.1021/acschemneuro.2c00544

Figure Lengend Snippet: Flow cytometry quantification of probe labeling. (a) Schematic of flow cytometry workflow. (b) DRD2-expressing 293T cells or untransduced 293T cells (negative control) are treated with a 100 nm probe, which is photo-cross-linked, and an Alexa Fluor 555 azide is then “clicked” onto the probe. The cells were then analyzed by flow cytometry. P values determined using two-way ANOVA in GraphPad; **** corresponds to P < 0.0001.

Article Snippet: Following blocking, the membrane was incubated with either 1 μg/mL Anti-Strep Tag II rabbit polyclonal antibody (Abcam, ab76949) or 2 μg/mL Anti-DRD2 rabbit polyclonal antibody (AbClonal, A12930) in TBST with 5% BSA overnight at 4 °C.

Techniques: Flow Cytometry, Labeling, Expressing, Negative Control

Journal: ACS Chemical Neuroscience

Article Title: Developing Photoaffinity Probes for Dopamine Receptor D 2 to Determine Targets of Parkinson’s Disease Drugs

doi: 10.1021/acschemneuro.2c00544

Figure Lengend Snippet: Photo-cross-linking of probes 5 and 7 to DRD2 visualized with Western blot. Lanes 1–5 correspond to samples with probe 5 at 100 nm, lanes 6–10 correspond to samples treated with probe 7 at 100 nm, and lanes 11–14 correspond to samples treated with negative control probe 16 . Lanes: 1, 6, and 11 are the anti-DRD2 antibody channel, lanes 2, 3, 7, 8, and 12 are fluorescence of the Alexa Fluor 555 clicked to the probes 5 , 7 , or 16 , lanes 4, 9, and 13 are the anti-Strep Tag antibody channel, and lanes 5, 10, and 14 are the overlaid channels for the respective probes.

Article Snippet: Following blocking, the membrane was incubated with either 1 μg/mL Anti-Strep Tag II rabbit polyclonal antibody (Abcam, ab76949) or 2 μg/mL Anti-DRD2 rabbit polyclonal antibody (AbClonal, A12930) in TBST with 5% BSA overnight at 4 °C.

Techniques: Western Blot, Negative Control, Fluorescence, Strep-tag

Journal: Molecular Neurobiology

Article Title: Electroacupuncture Alleviates Anxiety-Like Behaviors Induced by Chronic Neuropathic Pain via Regulating Different Dopamine Receptors of the Basolateral Amygdala

doi: 10.1007/s12035-022-02911-6

Figure Lengend Snippet: Expression of different DRs and EA-mediated regulation of these receptors in SNI mice. A and B Western blot images showing the protein levels and the quantification of DRD1 ( A ) and DRD2 ( B ) in the AMY 14 days after SNI surgery. ( n = 5–6 per group). C and D Western blot images showing the protein levels and the quantification of DRD1 ( C ) and DRD2 ( D ) in the AMY of SNI mice treated with EA. ( n = 6 per group). Data are expressed as the mean ± SEM. * p < 0.05 comparison of the two groups

Article Snippet: The membranes were blocked with 5% nonfat milk dissolved in TBST at room temperature for 1 h and incubated with the primary antibodies anti-DRD1 rabbit polyclonal antibody (1:1000, ab81296, Abcam) and anti-DRD2 rabbit polyclonal antibody (1:500, 55,084–1-AP, Proteintech) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Molecular Neurobiology

Article Title: Electroacupuncture Alleviates Anxiety-Like Behaviors Induced by Chronic Neuropathic Pain via Regulating Different Dopamine Receptors of the Basolateral Amygdala

doi: 10.1007/s12035-022-02911-6

Figure Lengend Snippet: The effects of EA and the DRD2 antagonist sulpiride on anxiety-like behaviors induced by microinjection of the DRD2 agonist quinpirole in the BLA. A Schematic of EA treatment and drug injection and behavioral testing. B Diagram showing the cannulation location (left) and a representative coronal section of the BLA from a mouse brain showing the cannula tip upon injection (right). C The effect of treatment in each group on the PWTs. ( n = 9 per group). D – F The effect of treatment in each group on anxiety-like behaviors in the EPM ( D ) and OFT ( E – F ). ( n = 7–9 per group). G Representative diagram showing tracked movement and activity heatmaps from the EPM. H Representative diagram showing tracked movement and activity heatmaps from the OFT. Data are expressed as the mean ± SEM. Tukey’s post hoc test: * p < 0.05 comparison of the two groups

Article Snippet: The membranes were blocked with 5% nonfat milk dissolved in TBST at room temperature for 1 h and incubated with the primary antibodies anti-DRD1 rabbit polyclonal antibody (1:1000, ab81296, Abcam) and anti-DRD2 rabbit polyclonal antibody (1:500, 55,084–1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Activity Assay

Journal: Molecular Neurobiology

Article Title: Electroacupuncture Alleviates Anxiety-Like Behaviors Induced by Chronic Neuropathic Pain via Regulating Different Dopamine Receptors of the Basolateral Amygdala

doi: 10.1007/s12035-022-02911-6

Figure Lengend Snippet: The effects of EA and the DRD2 antagonist sulpiride on pain-related allodynia and anxiety-like behaviors induced by SNI in mice. A Schematic of EA treatment and drug injection and behavioral testing. B Diagram showing the cannulation location (left) and a representative coronal section of the BLA from a mouse brain containing a cannula tip after injection (right). C The effect of treatment in each group on the PWTs. ( n = 9–10 per group). D – F The effect of treatment in each group on anxiety-like behaviors in the EPM ( D ) and OFT ( E – F ). ( n = 7–10 per group). G Representative diagram showing tracked movement and activity heatmaps from the EPM. H Representative diagram showing tracked movement and activity heatmaps from the OFT. Data are expressed as the mean ± SEM. Tukey’s post hoc test: + p < 0.05 compared with the sham + vehicle group; # p < 0.05 compared with the SNI + vehicle group; $ p < 0.05 compared with the SNI + sulpiride group; * p < 0.05 comparison between the two groups

Article Snippet: The membranes were blocked with 5% nonfat milk dissolved in TBST at room temperature for 1 h and incubated with the primary antibodies anti-DRD1 rabbit polyclonal antibody (1:1000, ab81296, Abcam) and anti-DRD2 rabbit polyclonal antibody (1:500, 55,084–1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Activity Assay