Journal: Brain, behavior, and immunity

Article Title: HIV-1 Tat protein enhances sensitization to methamphetamine by affecting dopaminergic function

doi: 10.1016/j.bbi.2017.05.004

Figure Lengend Snippet: Immunohistochemistry on paraffin embedded sections was utilized examine the protein distribution and levels of dopamine receptor D1 (A, B, C, D), dopamine receptor D2 (E, F, G, H), as well as of IBA-1 (I, J, K, L) in SAL TAT− (A, E, I), SAL TAT+ (B, F, J), METH TAT− (C, G, K), and METH TAT+ (D, H, L) mice. Representative positive cells in the 40× magnification images were labeled with a black arrow. (M) Normalized intensity density was calculated in ImageJ. Data are expressed as Mean ± SEM (n=5). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were blocked with 5g/l Casein (Sigma Aldrich) in PBS, containing 0.5g/l Thimerosal (Sigma Aldrich) and incubated with Iba-1 antibody (Wako Lab Chemicals, Richmond, VA), the anti-mouse DRD1 antibody (NLS43, Novus Biologicals, Littleton, CO), or anti mouse DRD2 (orb154598, Biorbyt, San Francisco, CA), each one diluted in Casein buffer.

Techniques: Immunohistochemistry, Labeling

Journal: Psychopharmacology

Article Title: Extracellular Dopamine, Acetylcholine, and Activation of Dopamine D1 and D2 Receptors after Selective Breeding for Cocaine Self-Administration in Rats

doi: 10.1007/s00213-017-4640-7

Figure Lengend Snippet: Counts of cells expressing c-Fos, D1R and D2R in different brain regions of LS and HS rats. Values indicate the number of cells per field, scored under blinded conditions, with data shown for five animals per condition.

Article Snippet: Sections were then incubated with sheep anti-c-Fos (1:500, CBL440, Millipore, Temecula, USA), combined with either mouse anti-Dopamine D1R (1:1000, NB110, Novus Biologicals, Littleton, USA) or rabbit anti-Dopamine D2R (1:500, AB5084P, Millipore, Temecula, USA) antibodies over 48 h in a humidi ed chamber at 4°.

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Distinct Transcriptomic Profiles of Cultured Anterior and Posterior Populations of Human Infant Scleral Fibroblasts: Including Dopamine Receptors

doi: 10.1167/iovs.66.5.29

Figure Lengend Snippet: D1-like receptor activity promotes the inhibitory effect of DA on cell contraction, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, impedes it. ( a – p ) The cell contraction rates at 24 hours for anterior ( a – h ) and posterior ( i – p ) scleral fibroblasts from HSF12 and HSF13 were measured following treatment with a range of DA receptor antagonists and agonists, administered 1 hour prior to the addition of DA. One experiment was conducted with three replicate wells for each compound tested, using two biological samples (HSF12 and HSF13; total n = 6). Results from the two cell lines were averaged and presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-way ANOVA).

Article Snippet: The antibodies used were as follows: Dopamine Receptor D1 Rabbit mAb (381747; Zen-Bioscience), DRD2 polyclonal antibody (55084-1-AP; Proteintech, Rosemont, IL, USA), Dopamine Receptor D3 Rabbit mAb (382983; Zen-Bioscience), DRD4 Rabbit polyclonal antibody (28094-1-AP; Proteintech), Dopamine Receptor D5 Rabbit pAb (820522; Zen-Bioscience), GAPDH (7E4) Mouse mAb (200306-7E4; Zen-Bioscience), Beta Tubulin Polyclonal antibody (10094-1-AP; Proteintech), Goat anti-Rabbit IgG(H&L) (HRP conjugate) (511203; Zen-Bioscience), and Goat anti-Mouse IgG (H&L) (HRP conjugate) (511103; Zen-Bioscience).

Techniques: Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Western blots for D1R, D2R and ß-actin in WT (n=ll) and EAAC1 −/− mice (n=8) show decreased levels of D1R (***p=2.7e-4), not D2R (p=0.51), in EAAC1 −/− mice. (B) Western blot analysis for DARPP-32, showing no significant difference in its expression between WT (n=15) and EAAC1 −/− mice (n=12, p=0.92). (C) Western blot analysis for pDARPP-32 T34 (WT (n=10), EAAC1 −/− (n=10), p=0.41), pDARPP-32 175 (WT (n=13), EAAC1 −/− (n=14), p=0.84), pDARPP-32 S97 (WT (n=13), EAAC1 −/− (n=13), p=0.98), pDARPP-32 S130 (WT (n=14), EAAC1 −/− (n=17), *p=0.024). (D) Pie chart representation of the phosphorylation distribution on the T34, T75, S97 and S130 sites of DARPP-32. The red curve highlights the S130 site, which shows increased phosphorylation in EAAC1 −/− mice. (E) As in (C), following data normalization by the average band intensity values measured in WT mice (pDARPP-32 T34 WT (n=10), EAAC1 −/− (n=10), p=0.46; pDARPP-32 T75 WT (n=13), EAAC1 −/− (n=14), p=0.72; pDARPP-32 S97 WT (n=13), EAAC1 −/− (n=13), p=0.89; pDARPP-32 5130 WT (n=14), EAAC1 −/− (n=15), **p=2.1e-3). Data in panels ?,?,? represent the band intensity ratio between the target protein and β-actin in samples from WT versus EAAC1 7 ' mice in the same blotting membrane.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Western blots for D1R, D2R and β-actin, in the presence of the mGluRI blockers LY367385 (50 μM) and MPEP (10 μM), show no significant difference in the expression of D1R and D2R (D1R: WT (n=9), EAAC1 −/− (n=9), p=0.35; D2R: WT (n=7), EAAC1 −/− (n=7), p=0.064). (B) mGluRI blockade induces a slight reduction in DARPP-32 expression between WT (n=9) and EAAC1 −/− mice (n=9, *p=0.029). (C) Western blot analysis for pDARPP-32 T34 (WT (n=5), EAAC1 −/− (n=6), p=0.98), pDARPP-32 T75 (WT (n=6), EAAC1 −/− (n=7), *p=0.017), pDARPP-32 S97 (WT (n=9), EAAC1 −/− (n=9), p=0.34), pDARPP-32 S130 (WT (n=12), EAAC1 −/− (n=9), **p=4.6e-3). (D) Pie chart representation of the phosphorylation distribution on the T34, T75, S97 and S130 sites of DARPP-32 in the presence of mGluRI blockers. The red curves highlight the T75 and S130 site, which show reduced phosphorylation in EAAC1 −/− mice. (E) As in (C), following data normalization by the average band intensity values measured in WT mice (WT (n=5), EAAC1 −/− (n=6), p=0.62), pDARPP-32 T75 (WT (n=9), EAAC1 −/− (n=7), ***p=1.0e-5), pDARPP-32 S97 (WT (n=9), EAAC1 −/− (n=9), p=0.16), pDARPP-32 S130 (WT (n=12), EAAC1 −/− (n=9), ***p=2.1e-4). Data in panels A,B,E represent the band intensity ratio between the target protein and β-actin measured in samples from EAAC1 −/− mice and normalized by analogous measures in samples from WT mice blotted in the same membrane.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Timeline of the experimental design. At P14-16, mice received a unilateral stereotaxic injection of hM3D(Gq). After one week, they started receiving daily I/P saline injections. At P28-30, they received I/P injections of CNO (5 mg/Kg). One hour after the CNO injections, they were video-monitored to examine their grooming behavior. Two hours after the CNO injections, they were sacrificed. Proteins for Western blot analysis were extracted from the control and injected striatum and from the adjacent cortices. (B) Left: mCherry expression in D1 Cre/+ mice. A significant increase in mCherry expression was detected only in the striatum of D1 Cre/+ mice (n=10, *p=0.040). Right: mCherry expression in A2A Cre/+ mice. A significant increase in mCherry expression was detected only in the striatum of A2A Cre/+ mice (n=23, *p=0.035). (C) Left: D1R and D2R expression in D1 Cre/+ mice (D1R: n=9, *p=0.013; D2R: n=8, p=0.35). The expression of D1R is significantly reduced in the injected striatum. Right: D1R and D2R expression in A2A Cre/+ mice (D1R: n=9, p=0.63; D2R: n=5, p=0.34). The expression of D1R and D2R is similar in the injected and non-injected striatum. (D) Left: hM3D(Gq) injection in D1 Cre/+ mice leads to increased pDARPP-32 S130 (pDARPP-32 T34 n=10, p=0.92; pDARPP-32 T75 n=11, p=0.18; pDARPP-32 S97 n=11, p=0.13, pDARPP-32 S130 n=11, **p=5.6e-3). Right: hM3D(Gq) injection in A2A Cre/+ mice leads to no change in pDARPP-32 (pDARPP-32 T34 n=9, p=0.75; pDARPP-32 T75 n=8, p=0.84; pDARPP-32 S97 n=8, p=0.31, pDARPP-32 S130 n=6, p=0.89). (E) Summary of the frequency (Frequency: Sham (n=25), D1 Cre/+ (n=39), Sham vs D1 Cre/+ **p=6.0e-3, A2A Cre/+ (n=49), Sham vs A2A Cre/+ p=0.28) and duration of grooming episodes in Sham and D1 Cre/+ mice injected with hM3D(Gq) (Duration: Sham (n=34), D1 Cre/+ (n=44), Sham vs D1 Cre/+ p=0.12, A2A Cre/+ (n=58), Sham vs A2A Cre/+ p=0.08). (F) Relationship between the frequency and duration of grooming episodes in Sham (left), D1 Cre/+ (middle) and A2A Cre/+ mice (right). The inset represents a density plot of the data, with blue areas corresponding to the duration and frequency of the most commonly observed grooming episodes.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Injection, Western Blot, Expressing