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    Alomone Labs dopamine d2r for co ip
    The effects of D1-D2 heteromer stimulation and inactivation on basal conditioned place preference. (A) Vehicle-conditioned rats did not exhibit a preference toward a particular chamber. D1-D2 heteromer stimulation by SKF 83959 (1.5 mg/kg, s.c.) induced conditioned place aversion (CPA) as the animals spent significantly less time in the drug paired chamber. (B) SKF 83959-induced CPA was abolished by pre-treatment by the D1-D2 heteromer selective disrupting peptide, TAT-D1, but not the control TAT-Sc peptide. (C) Inactivation of D1-D2 heteromer by TAT-D1 resulted in conditioned place preference (CPP) as the rats spent significantly more time in the drug paired chamber, not observed with the control TAT-Sc. (D) Representative western blots (inset) and histogram showing the amount of D1R co-immunoprecipitated with <t>D2R</t> from the NAc of rats treated with saline or SKF 83959. Pretreatment with TAT-D1 led to decreased co-immunoprecipitated receptors. An aliquot of each sample was used as a control for WB (input control). (E,F) The CPA induced by D1-D2 heteromer stimulation was abolished by Cdk5 inhibitor roscovitine pre-treatment (200 nmol, i.c.v, E ) or intra-accumbal injection (30 nmol, F ). (G) Representative western blot and histogram showing the density of Thr75-DARPP-32 phosphorylation (pT75) relative to GAPDH (as loading control). Data represent means ± SEM of n = 8–10 rats/group. ( * p
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    The effects of D1-D2 heteromer stimulation and inactivation on basal conditioned place preference. (A) Vehicle-conditioned rats did not exhibit a preference toward a particular chamber. D1-D2 heteromer stimulation by SKF 83959 (1.5 mg/kg, s.c.) induced conditioned place aversion (CPA) as the animals spent significantly less time in the drug paired chamber. (B) SKF 83959-induced CPA was abolished by pre-treatment by the D1-D2 heteromer selective disrupting peptide, TAT-D1, but not the control TAT-Sc peptide. (C) Inactivation of D1-D2 heteromer by TAT-D1 resulted in conditioned place preference (CPP) as the rats spent significantly more time in the drug paired chamber, not observed with the control TAT-Sc. (D) Representative western blots (inset) and histogram showing the amount of D1R co-immunoprecipitated with D2R from the NAc of rats treated with saline or SKF 83959. Pretreatment with TAT-D1 led to decreased co-immunoprecipitated receptors. An aliquot of each sample was used as a control for WB (input control). (E,F) The CPA induced by D1-D2 heteromer stimulation was abolished by Cdk5 inhibitor roscovitine pre-treatment (200 nmol, i.c.v, E ) or intra-accumbal injection (30 nmol, F ). (G) Representative western blot and histogram showing the density of Thr75-DARPP-32 phosphorylation (pT75) relative to GAPDH (as loading control). Data represent means ± SEM of n = 8–10 rats/group. ( * p

    Journal: Frontiers in Pharmacology

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

    doi: 10.3389/fphar.2017.00924

    Figure Lengend Snippet: The effects of D1-D2 heteromer stimulation and inactivation on basal conditioned place preference. (A) Vehicle-conditioned rats did not exhibit a preference toward a particular chamber. D1-D2 heteromer stimulation by SKF 83959 (1.5 mg/kg, s.c.) induced conditioned place aversion (CPA) as the animals spent significantly less time in the drug paired chamber. (B) SKF 83959-induced CPA was abolished by pre-treatment by the D1-D2 heteromer selective disrupting peptide, TAT-D1, but not the control TAT-Sc peptide. (C) Inactivation of D1-D2 heteromer by TAT-D1 resulted in conditioned place preference (CPP) as the rats spent significantly more time in the drug paired chamber, not observed with the control TAT-Sc. (D) Representative western blots (inset) and histogram showing the amount of D1R co-immunoprecipitated with D2R from the NAc of rats treated with saline or SKF 83959. Pretreatment with TAT-D1 led to decreased co-immunoprecipitated receptors. An aliquot of each sample was used as a control for WB (input control). (E,F) The CPA induced by D1-D2 heteromer stimulation was abolished by Cdk5 inhibitor roscovitine pre-treatment (200 nmol, i.c.v, E ) or intra-accumbal injection (30 nmol, F ). (G) Representative western blot and histogram showing the density of Thr75-DARPP-32 phosphorylation (pT75) relative to GAPDH (as loading control). Data represent means ± SEM of n = 8–10 rats/group. ( * p

    Article Snippet: AntibodiesThe primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit).

    Techniques: Conditioned Place Preference, Western Blot, Immunoprecipitation, Injection

    Evidence for the existence of dopamine D1-D2 receptor heteromer in rat NAc. (A) Proximity ligation assay (PLA) was used to visualize and detect D1R and D2R close proximity. (A1) A scheme depicts the PLA probes used in the present study. (A2–A4) Representative images of PLA signals (red dots) in neurons (nuclei stained by DAPI) in rat caudate putamen (CPu), nucleus accumbens core (NAc-core) and shell (NAc-shell) subregions. (A5) Graph representing the percent of neurons with a positive PLA signal. (A6) Representative image of PLA signals in neurons (nuclei stained by DAPI) in rat NAc-core using the second set of antibodies. (B) Representative images of immunohistochemistry using D1R antibody (D1R-Ab) or D2R antibody (D2R-Ab) directly conjugated to Alexa-488 or Alexa-568, respectively, in the NAc-shell. Direct confocal FRET analysis was performed, reflected by FRET efficiency (FRET E) and the distance between the dipoles, less than 10 nm (100 Å). (C) A representative close-up of a single MSN cell body from NAc showing D1R-D2R colocalization (left), D1-D2 heteromer FRET efficiency (center) and relative distance between receptors (right). (D,E) Histograms showing FRET E ratios (D) and distance (E) obtained from MSN cell bodies from NAc ( n = 24). Bars are 10 μm.

    Journal: Frontiers in Pharmacology

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

    doi: 10.3389/fphar.2017.00924

    Figure Lengend Snippet: Evidence for the existence of dopamine D1-D2 receptor heteromer in rat NAc. (A) Proximity ligation assay (PLA) was used to visualize and detect D1R and D2R close proximity. (A1) A scheme depicts the PLA probes used in the present study. (A2–A4) Representative images of PLA signals (red dots) in neurons (nuclei stained by DAPI) in rat caudate putamen (CPu), nucleus accumbens core (NAc-core) and shell (NAc-shell) subregions. (A5) Graph representing the percent of neurons with a positive PLA signal. (A6) Representative image of PLA signals in neurons (nuclei stained by DAPI) in rat NAc-core using the second set of antibodies. (B) Representative images of immunohistochemistry using D1R antibody (D1R-Ab) or D2R antibody (D2R-Ab) directly conjugated to Alexa-488 or Alexa-568, respectively, in the NAc-shell. Direct confocal FRET analysis was performed, reflected by FRET efficiency (FRET E) and the distance between the dipoles, less than 10 nm (100 Å). (C) A representative close-up of a single MSN cell body from NAc showing D1R-D2R colocalization (left), D1-D2 heteromer FRET efficiency (center) and relative distance between receptors (right). (D,E) Histograms showing FRET E ratios (D) and distance (E) obtained from MSN cell bodies from NAc ( n = 24). Bars are 10 μm.

    Article Snippet: AntibodiesThe primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit).

    Techniques: Proximity Ligation Assay, Staining, Immunohistochemistry