drd1 rabbit polyclonal antibody  (Proteintech)

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: Initial characterization of a transgenic mouse with overexpression of the human D 1 -dopamine receptor in the heart

doi: 10.1007/s00210-023-02901-y

Figure Lengend Snippet: Western blots. A A typical Western blot for the D 1 receptor in hearts from WT, D 1 -TG, and D 1 -KO after incubation with the D 1 -dopamine receptor antibody is seen. The corresponding band for the D 1 receptor (DRD1) is labelled with an arrow. As loading control, the Ponceau S-stained membrane is shown below. B Typical Western blots for regulatory proteins in hearts from WT and D 1 -TG after incubation with the D 1 -dopamine receptor agonist SKF 38393 are seen. Western blots depict the phosphorylation state of inhibitory subunit of troponin (P-TnI) with arrows. As a loading control, we assessed the protein expression of cardiac calsequestrin (CSQ) by cutting horizontally the lanes of the blot and incubating the lower and upper halves with different primary antibodies. M rainbow marker, LA left atrium, RA right atrium, VT ventricle

Article Snippet: First antibodies were rabbit polyclonal anti-calsequestrin (CSQ) antibody, #ab3516, Abcam, Cambridge, UK (diluted 1:20000), rabbit polyclonal anti-DRD1, #bs-1007R, Bioss, Woburn, MA, USA (dilution 1:500), and rabbit polyclonal anti-phospho-troponin I (P-TnI) antibody, #4004, Cell Signaling Technology Europe, Leiden, The Netherlands (diluted 1:5000).

Techniques: Western Blot, Incubation, Control, Staining, Membrane, Expressing, Marker

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: Initial characterization of a transgenic mouse with overexpression of the human D 1 -dopamine receptor in the heart

doi: 10.1007/s00210-023-02901-y

Figure Lengend Snippet: A Autoradiography. Representative images of autoradiography of D 1 -TG and WT mouse heart sections labeled with 5 nM [ 3 H]SCH23390 (total binding, TB) or 5 nM [ 3 H]SCH23390 + 10 M butaclamol (unspecific binding, UB). B In situ hybridization. Expression of D 1 -dopamine receptor mRNA detected by DIG-labelled human D 1 -dopamine receptor antisense probes (A-X) and DIG-labelled mouse D 1 -dopamine receptor antisense probes (a-l). DIG-labeled sense probes were used as controls. (A-H): Low magnification microscopic images, scale bar = 500 µm. mRNA expression of D 1 -dopamine receptor was not detected in WT mouse atria (A) and ventricles (C); whereas expression of human D 1 -dopamine receptor mRNA was detected in D 1 -Tg mouse atria (E) and ventricles including interventricular septum (G). (B, D, F, H) are corresponding control sections hybridized with DIG-labeled sense probes. (I-X): High magnification microscopic images, scale bar = 50 µm. No or very scarce and weak D 1 -dopamine mRNA expression was detected in WT mouse atria (I) and interventricular septum (K), while abundant human D 1 -dopamine mRNA expression was detected in atriums (M), interventricular septum (O), and ventricle walls (Q, S). (J, L, N, P, R, T) are corresponding control sections hybridized with DIG-labeled sense probes. D 1 -dopamine mRNA expression was also detected in human cardiomyocytes (U, W); while no signal was seen in corresponding control sections hybridized with DIG-labeled sense probes (V, X). (a-d): Low magnification microscopic images, scale bar = 500 µm. (e-h): High magnification microscopic images, scale bar = 50 µm. Expression of mouse D 1 -dopamine receptor mRNA was not detected in WT mouse atria (a, e) and ventricles (c, g); (b, d, f, h) are corresponding control sections hybridized with DIG-labeled sense probes. Low magnification images (i and j, scale bar = 500 µm) and high magnification images (k and l, scale bar = 50 µm) showed that the mouse antisense probe also detected human D 1 -dopamine mRNA expression in D 1 -TG mouse interventricular septum (i, k), while the mouse sense probe (control) did not detect any specific signal (j, l). C : Histology. Histological staining of myocardial sections with hematoxylin-eosin (HE) or Masson-Goldner trichome (MG) staining revealed no apparent abnormalities or signs of fibrosis, neither in WT nor in D 1 -TG. Scale bar = 100 µm. D : PCR. Scatter plots with means for the expression of exogenous transgenic D 1 -dopamine receptors (human-DRD1, left hand side) or endogenous mouse D 1 -dopamine receptors. Ordinates indicate the amount of the mRNA as inferred from the amplification cycles (Cq). *indicates significant differences between the mRNA from the whole heart of D 1 -TG versus WT

Article Snippet: First antibodies were rabbit polyclonal anti-calsequestrin (CSQ) antibody, #ab3516, Abcam, Cambridge, UK (diluted 1:20000), rabbit polyclonal anti-DRD1, #bs-1007R, Bioss, Woburn, MA, USA (dilution 1:500), and rabbit polyclonal anti-phospho-troponin I (P-TnI) antibody, #4004, Cell Signaling Technology Europe, Leiden, The Netherlands (diluted 1:5000).

Techniques: Autoradiography, Labeling, Binding Assay, In Situ Hybridization, Expressing, Control, Staining, Transgenic Assay, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Alteration of the Dopamine Receptors’ Expression in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant (Naked–Ataxia ( NAX )) Mouse

doi: 10.3390/ijms21082914

Figure Lengend Snippet: RNA sequencing data analysis in nax ( n = 5) and wild–type (wt) ( n = 6) mice. Data analysis shows dopamine receptor D1 ( Drd1, A ), Drd3 ( C ), and Drd4 ( D ) are significantly increased in the nax cerebellum. Although an increase in Drd2 ( B ) and Drd5 ( E ) is apparent, statistical significance was not reached. The data in the bar graph are presented as the mean ± SEM, and statistical analysis was performed using an unpaired t –test (* p < 0.05 and ** p < 0.01). RPKM: Reads Per Kilobase of transcript per Million mapped reads.

Article Snippet: D1: rabbit polyclonal anti–D1 dopamine receptor (TA328798, anti–Drd1, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA); D2: rabbit polyclonal anti–D2 dopamine receptor (TA328800, anti–Drd2, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor 2 (DR2); D3: rabbit polyclonal anti–D3 dopamine receptor (TA328800, anti–Drd3, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor 3 (DR3); D4: rabbit polyclonal anti–D4 dopamine receptor (TA321202, anti–DRD4, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor D4 (DRD4); and D5: rabbit polyclonal anti–D5 dopamine receptor (TA328802, anti–Drd5, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor 5 (DR5).

Techniques: RNA Sequencing Assay

Journal: International Journal of Molecular Sciences

Article Title: Alteration of the Dopamine Receptors’ Expression in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant (Naked–Ataxia ( NAX )) Mouse

doi: 10.3390/ijms21082914

Figure Lengend Snippet: Frontal sections of wt and nax mouse cerebella: DRD1 expression at P5 and P17. ( A ) Immunoperoxidase staining for DRD1 in the wt cerebellum on P5 shows the immunoreactivity in the Purkinje cell layer (Pcl) and white matter (wm) but not in the external germinal zone (egz) and granular layer (gl). ( B ) Immunoperoxidase staining for DRD1 in the nax cerebellum shows similar immunoreactivity as in the wt littermates at P5. ( C ) DRD1 immunostaining of the frontal sections of the wt cerebellum at P17 shows immunoreactivity in the Purkinje cell (PC) somata, the Pcl, and dendrites in the molecular layer (ml) and in the gl but not in the wm. ( D ) DRD1 immunostaining of the frontal sections of the nax cerebellum on P17 shows immunoreactivity in the PCs in the ml/Pcl. ( E ) Western blot analysis of whole cerebellar lysates revealed no significant differences in DRD1 expression between wt and nax cerebella at P5 and P17 (wt: n = 3 and nax : n = 3). The data in the bar graphs are presented as the means of three independent experiments ± SEM, and statistical analysis was performed using a two–way ANOVA. P; postnatal. Scale bars: 100 μm in A, B, C, and D.

Article Snippet: D1: rabbit polyclonal anti–D1 dopamine receptor (TA328798, anti–Drd1, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA); D2: rabbit polyclonal anti–D2 dopamine receptor (TA328800, anti–Drd2, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor 2 (DR2); D3: rabbit polyclonal anti–D3 dopamine receptor (TA328800, anti–Drd3, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor 3 (DR3); D4: rabbit polyclonal anti–D4 dopamine receptor (TA321202, anti–DRD4, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor D4 (DRD4); and D5: rabbit polyclonal anti–D5 dopamine receptor (TA328802, anti–Drd5, diluted 1:1000; OriGene Biotech Co., Rockville, MD, USA), produced against recombinant rat dopamine receptor 5 (DR5).

Techniques: Expressing, Immunoperoxidase Staining, Immunostaining, Western Blot

Journal: EMBO molecular medicine

Article Title: MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction.

doi: 10.1002/emmm.201201900

Figure Lengend Snippet: Figure 2. The effect of miR‐382 on the expression of DRD1 and DeltaFosB in rat NAc: p < 0.01, p < 0.001, Student’s t‐test. Source data is available for this figure in the Supporting Information. A. Seven-days’ alcohol injection (1 g/kg, i.p. bid) significantly increased the expression of DRD1 (p ¼ 0.00106) and DeltaFosB (p ¼ 0.00056) in rat NAc at the protein level. Values are mean SEM from 6 independent experiments (n ¼ 6), compared with that in vehicle-treated controls. B. Representative Western blots of DRD1 and DeltaFosB from animals treated with vehicle or alcohol. C. Alcohol injections significantly increased the expression of DRD1 (p ¼ 0.00022) and DeltaFosB (p ¼ 0.00047) in rat NAc at the mRNA level. Values are mean SEM from 6 independent experiments (n ¼ 6), compared with that in vehicle-treated controls. D. miR-382 expression in rat NAc was down-regulated by LNA-anti-miR-382 (Anti-miR-382) (p ¼ 0.00133), but was up-regulated by Ad-miR-382 (p ¼ 0.00102). Values are mean SEM from 4 independent experiments (n ¼ 4), compared with that in the controls [LNA-anti-miR-382 control (Anti-control), and Ad-GFP]. E. Left panel: view of injection site. Right panel: fluorescent image of GFP (green colour) in NAc at 5 days after microinjection of Ad-GFP. Injected Ad-GFP was limited to core region. Note: 40 mm slices, scale bar ¼ 500 mm. aca, anterior commissure; AcBC, nucleus accumbens core; AabSH, nucleus accumbens shell; LV, lateral ventricle. F. The expression of DRD1 (p ¼ 0.00057) and DeltaFosB (p ¼ 0.0004) in rat NAc was increased by LNA-anti-miR-382. Values are mean SEM from 6 independent experiments (n ¼ 6), compared with that in controls (Anti-control). G. Representative Western blots of DRD1 and DeltaFosB from animals treated with vehicle, anti-control or Anti-miR-382. H. Overexpression of miR-382 via Ad-miR-382 decreased the expression of DRD1 (p ¼ 0.00041) and DeltaFosB (p ¼ 0.00087) in rat NAc. Values are mean SEM from 6 independent experiments (n ¼ 6), compared with that in control (Ad-GFP). I. Representative Western blots of DRD1 and DeltaFosB from animals treated with vehicle, Ad-GFP or Ad-miR-382.

Article Snippet: A standard Western blot analysis was conducted using DeltaFosB rabbit monoclonal antibody (1:100 dilution; Cell Signalling) and DRD1 rabbit polyclonal antibody (1:1000 dilution; Proteintech) GAPDH antibody (1:5000 dilution; Cell Signalling) was used as a loading control. miRNA microarray analysis After treatment with alcohol and saline for 7 days, miRNAs were isolated from the NAc of rats using the mirVana miRNA isolation kit (Ambion, Inc.).

Techniques: Expressing, Injection, Western Blot, Control, Microinjection, Over Expression

Journal: EMBO molecular medicine

Article Title: MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction.

doi: 10.1002/emmm.201201900

Figure Lengend Snippet: Figure 3. Drd1 is a direct target gene of miR‐382 and is a regulator for the expression of DeltaFosB in cultured CAD cells: p < 0.01, p < 0.001, Student’s t‐test. Source data is available for this figure in the Supporting Information. A. Drd1 is a potential target gene of miR-382 predicted by computational analysis. B. The luciferase reporter construct, containing the putative miR-382 binding sequence from 30-UTR of rat Drd1 gene, was transfected into HEK293 cells with vehicle (Vehicle), an empty vector (pDNR-CMV), miR-382 (pmiR-31) or a control plasmid expressing an unrelated miRNA, miR-31 (pmiR-31). The construct with mutated fragment of the 30-UTR of Drd1 mRNA without the putative miR-382 binding sequences was used as the mutated control (mutated Drd1), pmiR- 382, but not pmiR-31 or pDNR-CMV, inhibited luciferase activity (p ¼ 1.17571599413E-6). Values are mean SEM from 6 independent experiments (n ¼ 6), compared with that in control (pDNR-CMV). In the mutated control group, the inhibitory effect of pmiR-miR-382 on luciferase activity disappeared. Values are mean SEM from 3 independent experiments (n ¼ 3), compared with that in control (pDNR-CMV). C. The expression of miR-382 in cultured CAD cells was modulated by LNA-anti-miR-382 (Anti-miR-382) and pre-miR-382. Vehicle and control oligos (oligo control) were used as controls. miR-382 expression was down-regulated by LNA-anti-miR-382 (p ¼ 5.08726615734E-5), but was up-regulated by pre-miR- 382 (p ¼ 1.69645597989E-5). Values are mean SEM from 5 independent experiments (n ¼ 5), compared with that in control (oligo control). D. At protein level, pre-miR-382 decreased the expression of DRD1 (p ¼ 2.44969469509E-5) and DeltaFosB (p ¼ 0.0008). In contrast, the expression of DRD1 (p ¼ 0.00089) and DeltaFosB (p ¼ 0.00149) was increased by LNA-anti-miR-382 (Anti-miR-382). Values are mean SEM from 5 independent experiments (n ¼ 5), compared with that in oligo control. E. Representative Western blots of DRD1 and DeltaFosB. F. pre-miR-382 decreased, whereas Anti-miR-382 increased the expression of Drd1 (p ¼ 0.00416 and 0.00039) and DeltaFosB (p ¼ 7.21292762637E-5 and 0.00027) at mRNA level. Values are mean SEM from 5 independent experiments (n ¼ 5), compared with that in oligo control. G. DRD1 protein was knocked-down by its siRNAs (DRD1-siRNA) (p ¼ 0.00137). Values are mean SEM from 3 independent experiments (n ¼ 3), compared with that in siRNA control. H. DeltaFosB protein was decreased via knocking-down of DRD1 by its siRNAs (p ¼ 0.00817). Values are mean SEM from 3 independent experiments (n ¼ 3), compared with that in siRNA control.

Article Snippet: A standard Western blot analysis was conducted using DeltaFosB rabbit monoclonal antibody (1:100 dilution; Cell Signalling) and DRD1 rabbit polyclonal antibody (1:1000 dilution; Proteintech) GAPDH antibody (1:5000 dilution; Cell Signalling) was used as a loading control. miRNA microarray analysis After treatment with alcohol and saline for 7 days, miRNAs were isolated from the NAc of rats using the mirVana miRNA isolation kit (Ambion, Inc.).

Techniques: Expressing, Cell Culture, Luciferase, Construct, Binding Assay, Sequencing, Transfection, Plasmid Preparation, Control, Activity Assay, Western Blot

Journal: EMBO molecular medicine

Article Title: MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction.

doi: 10.1002/emmm.201201900

Figure Lengend Snippet: Figure 4. Overexpression of miR‐382 is sufficient to inhibit alcohol‐induced up‐regulation of DRD1 and DeltaFosB in rat NAc: p < 0.01, p < 0.001, ##p < 0.001, Student’s t‐test. Source data is available for this figure in the Supporting Information. At 3 days before alcohol administration, 4 ml of vehicle, Ad-GFP or Ad-miR-382 (1 109 pfu/ml) was infused into the NAc of rats. Then, the animals were divided into the following groups: Vehicle-treated rats without alcohol administration (Vehicle); Ad-GFP- treated rats without alcohol administration (Ad-GFP); Ad-GFP-treated rats with alcohol administration (Alcohol þ Ad-GFP) and Ad-miR-382-treated rats with alcohol administration (Alcohol þ Ad-miR-382). Severn days later, the rat NAc were isolated. A. The successful modulation of miR-382 expression by Ad-miR-382. Alcohol decreased the expression of miR-382 in rat NAc (p ¼ 0.00076). Values are mean SEM from 3 independent experiments (n ¼ 3), Alcohol þ Ad-GFP compared with that in Ad-GFP control. Ad-miR-382 increased the expres- sion of miR-382 in NAc of rats with alcohol administration (p ¼ 0.00079). Values are mean SEM from 3 independent experiments (n ¼ 3), Alcohol þ Ad-miR-382 compared with that in Alcohol þ Ad-GFP control. B. Representative Western blots in rat NAc from different treatments. C. Downregulation of DRD1 and DeltaFosB via over- expression of miR-382 in NAc. Alcohol adminis- tration increased the expression of DRD1 (p ¼ 0.00616) and DeltaFosB (p ¼ 0.00087). Values are mean SEM from 5 independent experiments (n ¼ 5), Alcohol þ Ad-GFP compared with that in Ad-GFP control. Overexpression of miR- 382 via Ad-miR-382 prevented alcohol-induced up- regulation of DRD1 (##p ¼ 0.0002) and DeltaFosB (##p ¼ 0.00012) in rat NAc. Values are mean SEM from 5 independent experiments (n ¼ 5), Alcohol þ Ad-miR-382 compared with that in Alcohol þ Ad-GFP control.

Article Snippet: A standard Western blot analysis was conducted using DeltaFosB rabbit monoclonal antibody (1:100 dilution; Cell Signalling) and DRD1 rabbit polyclonal antibody (1:1000 dilution; Proteintech) GAPDH antibody (1:5000 dilution; Cell Signalling) was used as a loading control. miRNA microarray analysis After treatment with alcohol and saline for 7 days, miRNAs were isolated from the NAc of rats using the mirVana miRNA isolation kit (Ambion, Inc.).

Techniques: Over Expression, Isolation, Expressing, Control, Western Blot

Journal: EMBO molecular medicine

Article Title: MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction.

doi: 10.1002/emmm.201201900

Figure Lengend Snippet: Figure 5. Overexpression of miR‐382 is sufficient to inhibit the voluntary intake of and the preference for alcohol in rats: the animals (total n ¼ 24) under the intermittent access two‐bottle choice drinking paradigm were randomly divided into three groups which received infusion of Ad‐miR‐382, control adenovirus Ad‐GFP or vehicle (saline), respectively, into the NAc. Seven days later, the rats were allowed to resume ethanol consumption under the same drinking paradigm. The successful up-regulation of miR-382 via Ad-miR-382 was verified by qRT-PCR at 7 days after drinking (Supporting Information Fig S1). The successful modulation of DRD1 and DeltaFosB via Ad-miR-382 was verified by Western blot analysis at 7 days after drinking (Supporting Information Fig S2). A. The voluntary intake of alcohol was reduced via Ad-miR-382 at all points (7 days: p ¼ 0.00039; 9 days: p ¼ 0.00046; 11 days: p ¼ 0.00057; 13 days: p ¼ 0.00026; 17 days: p ¼ 0.00018; 19 days: p ¼ 0.00098; 21 days: p ¼ 0.013; 23 days: p ¼ 0.0035). B. Ad-miR-382 decreased the preference for alcohol at all points (7 days: p ¼ 0.0037; 9 days: p ¼ 0.0041; 11 days: p ¼ 0.0029; 13 days: p ¼ 0.0015; 17 days: p ¼ 0.0088; 19 days: p ¼ 0.0068; 21 days: p ¼ 0.018; 23 days: p ¼ 0.0029). C. Ad-miR-382 did not alter the water consumption. D. Ad-miR-382 did not alter the total fluid intake. Values are mean SEM from 8 independent experiments (n ¼ 8), compared with that in Ad-GFP control. p < 0.05, p < 0.01 and p < 0.001, two-way ANOVA with repeated measure.

Article Snippet: A standard Western blot analysis was conducted using DeltaFosB rabbit monoclonal antibody (1:100 dilution; Cell Signalling) and DRD1 rabbit polyclonal antibody (1:1000 dilution; Proteintech) GAPDH antibody (1:5000 dilution; Cell Signalling) was used as a loading control. miRNA microarray analysis After treatment with alcohol and saline for 7 days, miRNAs were isolated from the NAc of rats using the mirVana miRNA isolation kit (Ambion, Inc.).

Techniques: Over Expression, Control, Saline, Quantitative RT-PCR, Western Blot

Journal: EMBO molecular medicine

Article Title: MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction.

doi: 10.1002/emmm.201201900

Figure Lengend Snippet: Figure 6. Overexpression of miR‐382 influences responses of MSNs in NAc slices to DRD1 activation: 4 ml of vehicle, Ad‐GFP or Ad‐miR‐382 (1 109 pfu/ ml) was infused into the NAc of rats. Severn days later, the NAc were isolated. A‐C. The successful modulation of miR-382 expression by Ad-miR-382. Ad-miR-382 increased the expression of miR-382 in NAc of rats (p ¼ 0.00125). Values are mean SEM from 3 independent experiments (n ¼ 3), compared with that in Ad-GFP control. p < 0.01, Student’s t-test. Sample voltage traces in response to current injections (inset) from a MSN in acute brain slices of rats which received infusion of saline (B) and Ad-miR-382 (C), respectively. While the application of 1 mM SKF38393, the DRD1 agonist increased the firing rate of the MSN of rats that received saline injection, this was not seen in rats received Ad-miR-382 injection. D. Summary graphs showing that Ad-miR-382 NAc injection attenuated the firing rate of MSNs–induced by 1 mM SKF38393 (p ¼ 0.00654). Values are mean SEM from 4 independent experiments (n ¼ 4), compared with that in Ad-GFP control. p < 0.01, two-way ANOVA.

Article Snippet: A standard Western blot analysis was conducted using DeltaFosB rabbit monoclonal antibody (1:100 dilution; Cell Signalling) and DRD1 rabbit polyclonal antibody (1:1000 dilution; Proteintech) GAPDH antibody (1:5000 dilution; Cell Signalling) was used as a loading control. miRNA microarray analysis After treatment with alcohol and saline for 7 days, miRNAs were isolated from the NAc of rats using the mirVana miRNA isolation kit (Ambion, Inc.).

Techniques: Over Expression, Activation Assay, Isolation, Expressing, Control, Saline, Injection