rabbit anti cb 1 r antibody  (Cayman Chemical)

Journal: Molecular Medicine

Article Title: Progressive endocannabinoid system dysregulation in autosomal dominant polycystic kidney disease

doi: 10.1186/s10020-026-01457-w

Figure Lengend Snippet: ECS components are progressively dysregulated in human ADPKD kidney tissue. a Microarray analysis ( GSE7869 ) of human kidney tissue shows stepwise increases in CNR1 transcript from healthy cortex to minimally cystic (PKDm) and fully cystic (PKD) ADPKD tissue, with corresponding reductions in AEA-metabolizing enzymes NAPEPLD and FAAH . b Single-nucleus RNA-sequencing (snRNA-seq) analysis of human ADPKD kidneys ( n = 8) versus healthy controls ( n = 5) demonstrate consistent CNR1 upregulation and marked downregulation of NAPEPLD and FAAH , while 2-AG-metabolizing enzymes remain largely unchanged. c snRNA-seq analysis of diabetic kidney disease (DKD; n = 5 patients; controls n = 6) reveals minimal alterations in CNR1 and ECS-metabolizing enzymes. d Gene expression analysis by qPCR confirms CNR1 upregulation in human ADPKD kidney tissue ( n = 17) versus non-cystic nephrectomy controls ( n = 5), with concurrent changes in ECS enzyme transcription. e-i eCB quantification by liquid chromatography-tandem mass spectrometry (LC–MS/MS) reveals significant depletion of tissue anandamide (AEA); e , N -oleoylethanolamine (OEA); f , 2-arachidonoylglycerol (2-AG); h , and arachidonic acid (AA); i , while N -palmitoylethanolamine (PEA); g remains unchanged. j-l Western blot analysis shows substantial inter-individual variability in CB 1 R protein levels without significant difference between ADPKD ( n = 6) and control kidneys ( n = 4), but significant reductions in DAGLα/β, NAPEPLD, MGLL and FAAH protein. Western blots were normalized to total proteins. Data represent mean ± SEM. Statistics of control versus PKD represented by * and control versus PKDm by #. Statistical significance assessed by Mann–Whitney U test or unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Sections were stained with rabbit anti-CB 1 R antibody (1:200, 10006590, Cayman) followed by a goat anti-rabbit HRP conjugate (ImmPRESSTM, Vector laboratories).

Techniques: Microarray, RNA Sequencing, Gene Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, MANN-WHITNEY

Journal: Molecular Medicine

Article Title: Progressive endocannabinoid system dysregulation in autosomal dominant polycystic kidney disease

doi: 10.1186/s10020-026-01457-w

Figure Lengend Snippet: Temporal endocannabinoid system dysregulation during Pkd1 RC/RC disease progression. a-g Quantitative PCR analysis of ECS-related genes across disease stages (9 and 12 months) showing progressive upregulation of Cnr1 a and Cnr2 b transcripts, with corresponding changes in ECS metabolic enzymes: Dagla c , Daglb d , Napepld e , Mgll f , and Faah g . h-n Western blot analysis and quantification of ECS proteins across disease stages, demonstrating sustained CB 1 R protein elevation at 9 and 12 months h, i , stable 2-AG-related enzyme proteins (DAGLα, DAGLβ, MGLL; h, j, k, m ), and stage-specific changes in AEA-related enzymes including elevated NAPEPLD at 12 months ( h, l ) and reduced FAAH at 9 months ( h, n ). Data represent mean ± SEM from WT ( n = 5) and Pkd1 RC/RC ( n = 6–7) per group per timepoint. Statistical analysis: unpaired t -test comparing Pkd1 RC/RC to age-matched WT. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus WT

Article Snippet: Sections were stained with rabbit anti-CB 1 R antibody (1:200, 10006590, Cayman) followed by a goat anti-rabbit HRP conjugate (ImmPRESSTM, Vector laboratories).

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Western Blot

Journal: bioRxiv

Article Title: Cannabinoid receptor 1 positive allosteric modulator ZCZ011 shows differential effects on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice

doi: 10.1101/2024.05.10.593514

Figure Lengend Snippet: CB 1 R and CB 2 R expression levels were assessed in the prefrontal cortex, striatum, and cerebellum for vehicle- and ZCZ011-treated Tat(–) and Tat(+) mice via Western blot analysis. Data were normalized to the housekeeping protein GAPDH. ( A ) Representative immunoblots for CB 1 R, CB 2 R, and GAPDH for all groups used in the study. ( B ) In the prefrontal cortex, no changes were observed in CB 1 R expression. In striatum and cerebellum, a main effect of sex was seen with females displaying higher levels of CB 1 R as compared to males. Additionally, ZCZ011 differentially affected CB 1 R expression based on genotype in the striatum and cerebellum. ( C ) In the prefrontal cortex and striatum, a main effect of sex was seen with males displaying higher level of CB 2 R in the prefrontal cortex and females displaying higher level of CB 2 R in the striatum. Further, Tat expression increased CB 2 R in the prefrontal cortex. In the cerebellum, no changes were observed in CB 2 R expression. All data are expressed as mean ± the standard error of the mean (SEM). Statistical significance was assessed by ANOVAs; λ p ≤ 0.05 main effect of sex; α p ≤ 0.005 main effect of genotype; φ p ≤ 0.05 main drug x genotype interaction; δ p ≤ 0.05 main genotype x sex interaction. Separate ANOVAs for females and males; ι p ≤ 0.05 genotype effect for females. Follow-up Tukey’s post hoc tests; * p ≤ 0.05. ZCZ011 dose = 10 mg/kg. N = 32(16f); AU, arbitrary unit.

Article Snippet: Primary antibodies used in this study were, anti-CB 1 R (rabbit polyclonal; Proteintech, Cat# 17978-1-AP, 1:1000 dilution), anti-CB 1 R (rabbit polyclonal; AbClonal, Cat# A1762, 1:1000 dilution), anti-FAAH (mouse monoclonal; Abcam, Cat# ab54615, 1:1000 dilution), and anti-MAGL (rabbit polyclonal; Abcam, Cat# ab24701, 1:1000 dilution).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

doi: 10.1371/journal.pone.0029239

Figure Lengend Snippet: A , Basal [ 35 S]GTPγS binding was measured in cortical membranes from wild-type and DOR −/− mice. Membranes from cortices were prepared as described – , treated with vehicle or 1 µM SR141716 (SR) for 1 hour and subjected to [ 35 S]GTPγS binding as described in . Basal [ 35 S]GTPγS binding/10 µg protein in vehicle treated membranes is taken as 100%. Data represent Mean ± SEM (n = 3 individual animals in triplicate). Statistically significant differences between vehicle and SR141716 treatment are indicated *p<0.05, (t test). B , [ 35 S]GTPγS binding assay in cortical membranes from wild-type and DOR −/− mice. Membranes were treated with increasing concentrations of the CB 1 R agonist Hu210 (0–1 µM) and [ 35 S]GTPγS binding was measured as described in . EC 50 and E max values were calculated using GraphPad Prism software. Data represent Mean ± SEM (n = 3 individual animals in triplicate). *p<0.05; **p<0.01 for DOR−/− vs wild-type (t test). C , Localization of endogenous CB 1 R and DOR in mouse primary cortical cells, 14DIV. Cells fixed with 4%PFA in PBS and permeablized with 0.1% Triton, were immunostained with the goat polyclonal anti-CB 1 R(N-terminal) antibody (1∶500; green) and rat polyclonal anti-DOR antibody (1∶500; red) and visualized using Alexa 488-conjugated anti-goat (1∶1000) and Alexa 594-conjugated anti-rat (1∶1000) secondary antibodies using confocal microscopy as described in . Representative figure from 3 independent experiments shown.

Article Snippet: Monoclonal anti-phosphoERK, polyclonal anti-ERK, monoclonal anti-myc, polyclonal anti-phosphoDOR(S363), monoclonal anti-phosphoSTAT3 (Ser-727), polyclonal anti-phospho-p90rsk, polyclonal anti-STAT3, polyclonal anti-phosphop70S6K, polyclonal anti-BAD, polyclonal anti-lamin A/C and monoclonal anti-phosphoBAD antibodies were from Cell Signaling Technology Inc. Rabbit anti C-terminal CB 1 R antibody was from Cayman Chemicals.

Techniques: Binding Assay, GTPγS Binding Assay, Software, Confocal Microscopy

Journal: PLoS ONE

Article Title: Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

doi: 10.1371/journal.pone.0029239

Figure Lengend Snippet: A , Lysates (100–200 µg) from N2A CB1R and N2A CB1R DOR cells were subjected to immunoprecipitation with 1 µg of anti-CB 1 R (C-terminal) antibody, the immunoprecipitates were resolved on 10% SDS-PAGE and probed for the presence of myc-DOR using mouse monoclonal anti-myc antibody (1∶1000) and for CB 1 R using rabbit polyclonal anti-CB 1 R (C-terminal) antibody (1∶500) as described in . IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. B , CB 1 R-DOR complexes exhibit greater interaction with AP-2 than AP-3. Lysates (100–200 µg) from N2A CB1R and N2A CB1R DOR cells were subjected to immunoprecipitation using 1 µg of an anti-CB 1 R (C-terminal) antibody as described in . The immunoprecipitates were resolved on 10% SDS-PAGE and probed for the presence of AP-3 (1∶1000), AP-2 (1∶1000) and CB 1 R (C-term) (1∶500) using specific antibodies as described in . IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. C , Localization of endogenous CB 1 R in N2A CB1R and of CB 1 R and DOR in N2A CB1R DOR cells. Cells fixed with 4%PFA in PBS and permeablized with 0.1% Triton, were stained with the rabbit polyclonal anti-CB 1 R (C-terminal) antibody (1∶500; green) and the mouse monoclonal anti-myc antibody (1∶1000; red) and visualized using Alexa 488-coupled anti-rabbit or Alexa 594-coupled anti-mouse secondary antibodies (1∶1000) using confocal microscopy as described in . Representative of 3 independent experiments shown. D , Cell surface staining of endogenous CB 1 R and stably expressed DOR in N2A CB1R DOR cells. N2A CB1R and N2A CB1R DOR cells were stained with a goat polyclonal anti-CB1R (N-terminal) antibody (1∶500) and mouse monoclonal anti-myc antibodies (1∶1000) prior to fixation of the cells to label cell surface receptors, as described . After fixation, cells were visualized with Alexa 594-coupled anti-goat and Alexa 488-coupled anti-mouse secondary antibodies (1∶1,000) using confocal microscopy as described in . Representative of 3 independent experiments shown.

Article Snippet: Monoclonal anti-phosphoERK, polyclonal anti-ERK, monoclonal anti-myc, polyclonal anti-phosphoDOR(S363), monoclonal anti-phosphoSTAT3 (Ser-727), polyclonal anti-phospho-p90rsk, polyclonal anti-STAT3, polyclonal anti-phosphop70S6K, polyclonal anti-BAD, polyclonal anti-lamin A/C and monoclonal anti-phosphoBAD antibodies were from Cell Signaling Technology Inc. Rabbit anti C-terminal CB 1 R antibody was from Cayman Chemicals.

Techniques: Immunoprecipitation, SDS Page, Staining, Confocal Microscopy, Stable Transfection

Journal: PLoS ONE

Article Title: Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

doi: 10.1371/journal.pone.0029239

Figure Lengend Snippet: A , [ 35 S]GTPγS binding assay in membranes from N2A CB1R and N2A CB1R DOR cells. Membranes (10 µg) were treated with indicated concentrations of the CB 1 R agonist Hu210. [ 35 S]GTPγS binding was measured as described in . EC 50 and E max values were calculated using GraphPad Prism software. Data represent Mean ± SEM (n = 3 independent experiments in triplicate). B , Dose-response of Hu210-mediated ERK phosphorylation in N2A CB1R and N2A CB1R DOR cells. Starved N2A CB1R and N2A CB1R DOR cells seeded in 24 well-plates were treated with indicated concentrations of Hu210 for 5 minutes. Cell lysates (30 µg protein) were analyzed by Western blotting and probed for the levels of pERK (1∶1000) and ERK (1∶1000) as described in . IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). EC 50 and E max values were calculated using GraphPad Prism software. Data represent Mean ± SEM (n = 3 independent experiments). *p<0.05 for N2A CB1R DOR vs N2A CB1R (t test). C , Effect of DOR down-regulation on ERK phosphorylation. F11 cells transduced with the DOR shRNA expressing lentivirus were starved for 4–6 h and treated with Hu210 (100 nM) for 5 min. Cell lysates (30 µg protein) were analyzed by Western blotting and probed for the levels of pERK (1∶1000) and ERK (1∶1000) as described in . IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Data from 3 independent experiments is shown. *p<0.05 (t test). D , Examination of arrestin3 interaction with CB 1 R after Hu210 treatment. N2A CB1R and N2A CB1R DOR, starved for 4 hours were stimulated with 100 nM Hu210 for 5 minutes and cell lysates prepared as described in . Lysates (30 µg protein) were subjected to either Western blotting using rabbit anti-CB1R (C-terminal 1∶500) and mouse anti-arrestin 3 antibodies (1∶500) or to immunoprecipitation using 1 µg of agarose-coupled anti-CB 1 R (C-terminal) antibody. Immunoprecipitates were probed for arrestin3 levels by Western blot using the mouse anti-arrestin 3 antibody. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. E , Effect of Hu210 on arrestin recruitment. U2OS cells co-expressing ProLink/Enzyme Donor (PK)-tagged DOR and the Enzyme Activator (EA)-tagged arrestin3 fusion protein without or with CB 1 R were treated with indicated concentrations of Hu-210. Arrestin3 recruitment was determined using the PathHunter Detection Kit as described in . Data represent Mean ± SEM (n = 4). F , Effect of PLC inhibitor (U73122) on DOR phosphorylation at serine 363 after Hu210 treatment. N2A CB1R DOR cells were starved for 4–6 hours, and incubated with vehicle (DMSO) or U73122 (1 µM) for 30 minutes, then stimulated with 100 nM Hu210 for 5 minutes. Cell lysates (30 µg protein) were subjected to Western blotting using rabbit polyclonal phosphoDOR Ser 363 (1∶1000), mouse monoclonal anti-myc (1∶1000) antibodies and IR Dye 680 anti-rabbit and IR Dye 800 anti-mouse secondary antibodies (1∶10,000) as described in . Data represent Mean ± SEM (n = 3).

Article Snippet: Monoclonal anti-phosphoERK, polyclonal anti-ERK, monoclonal anti-myc, polyclonal anti-phosphoDOR(S363), monoclonal anti-phosphoSTAT3 (Ser-727), polyclonal anti-phospho-p90rsk, polyclonal anti-STAT3, polyclonal anti-phosphop70S6K, polyclonal anti-BAD, polyclonal anti-lamin A/C and monoclonal anti-phosphoBAD antibodies were from Cell Signaling Technology Inc. Rabbit anti C-terminal CB 1 R antibody was from Cayman Chemicals.

Techniques: GTPγS Binding Assay, Binding Assay, Software, Western Blot, Transduction, shRNA, Expressing, Immunoprecipitation, Incubation

Journal: PLoS ONE

Article Title: Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

doi: 10.1371/journal.pone.0029239

Figure Lengend Snippet: A , Hu210-treated N2A CB1R DOR cells exhibit increased survival compared to N2A CB1R cells. N2A CB1R or N2A CB1R DOR cells were treated with 1 µM Hu210 for the indicated days and survival measured by trypan blue exclusion as described in . Data represent Mean ± SEM (n = 4 in triplicate). B , Hu210-treated N2A CB1R DOR cells exhibit lower apoptosis as compared to N2A CB1R cells. Apoptosis of N2A CB1R or N2A CB1R DOR treated for 3 or 8 days with 1 µM Hu210 was measured using caspase-3 activity assay as described in . Data represent Mean ± SEM (n = 4 in triplicate); ***p<0.001 N2A CB1R DOR vs N2A CB1R (t test). C , CB 1 R antagonist treatment decreases neuronal survival of striatal neurons from wild-type but not DOR−/− mice. Primary striatal neurons from wild-type or from DOR−/− mice were prepared as described in . The CB 1 R antagonist AM251 (10 µM) was added to the growth media at DIV7 and cellular viability assessed at DIV10 as described in . Data represent Mean ± SEM (n = 2–4) D–E , A schematic of the signaling pathways emanating from CB 1 R in N2A CB1R ( D ) and N2A CB1R DOR ( E ) cells. Activation of CB 1 R in N2A CB1R DOR cells leads to differential activation of signaling molecules and phosphorylation of ERK substrates.

Article Snippet: Monoclonal anti-phosphoERK, polyclonal anti-ERK, monoclonal anti-myc, polyclonal anti-phosphoDOR(S363), monoclonal anti-phosphoSTAT3 (Ser-727), polyclonal anti-phospho-p90rsk, polyclonal anti-STAT3, polyclonal anti-phosphop70S6K, polyclonal anti-BAD, polyclonal anti-lamin A/C and monoclonal anti-phosphoBAD antibodies were from Cell Signaling Technology Inc. Rabbit anti C-terminal CB 1 R antibody was from Cayman Chemicals.

Techniques: Caspase-3 Activity Assay, Activation Assay