Journal: Lipids

Article Title: Fatty Acid Binding Protein-1 (FABP1) and the Human FABP1 T94A Variant: Roles in the Endocannabinoid System and Dyslipidemias.

doi: 10.1007/s11745-016-4155-8

Figure Lengend Snippet: Fig. 4 Schematic of FABP1’s role in endocannabinoid (AEA, 2-AG) trafficking and function in primary hepatocytes. By binding ananda- mide (AEA) and 2-arachidonoylglycerol (2-AG), FABP1 may influ- ence key aspects of the hepatic endocannabinoid system: (a) FABP1 may facilitate AEA and 2-AG release/solubilization into the cytosol after their enzymatic cleavage/synthesis from plasma membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase α and β (DAGLα and DAGLβ). FABP1 may or may not facilitate bound AEA and 2-AG for transport/efflux across the plasma membrane and activation of CB receptors on the exofacial leaflet and/or intracellular sites for degra- dation/hydrolysis. FABP1 is known to enhance the cytosolic transport of other bound ligands [5, 46, 52, 141, 251]. (b) FABP1 may facili- tate the reuptake of AEA and 2-AG from the plasma membrane after these lipophilic ligands cross the plasma membrane by diffusion or via G protein-coupled cannabinoid receptor 1 (CB1). Reuptake of 2-AG may also occur via G protein-coupled endocannabinoid recep- tor (CB2). CB2 is expressed only in embryonic liver and in diseased conditions such as fatty liver [252, 253]. CB1 and CB2 activation has been linked to diet-induced hepatic steatosis, primary biliary cir- rhosis, chronic hepatitis, and alcoholic liver [101]. CB1 activation in mice enhances lipogenesis through the sterol regulatory element binding protein-1c (SREBP1c) pathway which induces transcrip- tion of multiple genes in lipogenesis such as SREBP1c itself, acyl CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl CoA desaturase (SCD1) [132]. (c) Upon FABP1-mediated release of plasma membrane and/or CB receptor-bound AEA or 2-AG into the cytosol, FABP1 may transport the bound AEA (and possibly 2-AG) into the nucleus for PPARα activation. Although ananda- mide has been shown to bind PPARα and enhance PPARα activation [135], liver fat accumulation in hepatic steatosis results in decreased LCFA oxidation, likely through saturation and/or inhibition of the PPARα pathway by the increasing fat load or damage by inflamma- tion [101]. (d) FABP1 may also transport the bound AEA to the deg- radative enzyme fatty acid amide hydrolase (FAAH) localized with smooth endoplasmic reticulum, mitochondria, lipid droplets, and more rarely at the cell membrane [142–144] by a process analogous to that established for other FABP family members (FABP3, 5, 7) in brain [106–110, 144]. In human liver FABP1 may also transport AEA to/from lysosomes, where it is degraded by N-acylethanolamide acid amide hydrolase (NAAA) [143, 145]. Finally, FABP1 may transport bound 2-AG for degradation by monoacylglycerol lipase (MAGL), an enzyme found at lower levels in liver than brain or other tissues where it is localized diffusely in cytosol and less so in membranes without overall compartmental preference [142]

Article Snippet: ACC Acetyl-CoA carboxylase ACOX1 Acyl-CoA oxidase 1, palmitoyl AEA N-Arachidonoylethanolamide (anandamide) 2-AG 2-Arachidonoylglycerol ALB Albumin ARA C20:4n-6 arachidonic acid CB1 Cannabinoid receptor-1 CB2 Cannabinoid receptor-2 CPT1A Carnitine palmitoyl transferase IA, liver In conjunction with the Lipids 50th Anniversary Symposium, sponsored by Avanti Polar Lipids, at the 106th AOCS Annual Meeting.

Techniques: Binding Assay, Clinical Proteomics, Membrane, Activation Assay, Diffusion-based Assay, Inhibition

Journal: Lipids

Article Title: Fatty Acid Binding Protein-1 (FABP1) and the Human FABP1 T94A Variant: Roles in the Endocannabinoid System and Dyslipidemias.

doi: 10.1007/s11745-016-4155-8

Figure Lengend Snippet: Fig. 6 Human FABP1 T94A variant expression induces transcrip- tion of enzymes and receptors in the endocannabinoid system. Pri- mary human hepatocytes were cultured as described previously [63] followed by determination of mRNA levels encoding the human N-acylphosphatidylethanolamide phospholipase-D (NAPE-PLD), diacylglycerol lipase-α (DAGLα), fatty acid amide hydrolase-1 (FARAH1), fatty acid amide hydrolase (FARAH2), monoacylglyc- erol lipase (MAGL), and cannabinoid receptor-1 (CB1) similarly as for other human mRNA transcripts [213–215]. Values are expressed as the fold change in the ratio of respective mRNA in human FABP1 T94A variant (T94A)/mRNA in wild-type human FABP1. Data are the mean ± SEM (n = 7); *p < 0.05 for T94A vs WT

Article Snippet: ACC Acetyl-CoA carboxylase ACOX1 Acyl-CoA oxidase 1, palmitoyl AEA N-Arachidonoylethanolamide (anandamide) 2-AG 2-Arachidonoylglycerol ALB Albumin ARA C20:4n-6 arachidonic acid CB1 Cannabinoid receptor-1 CB2 Cannabinoid receptor-2 CPT1A Carnitine palmitoyl transferase IA, liver In conjunction with the Lipids 50th Anniversary Symposium, sponsored by Avanti Polar Lipids, at the 106th AOCS Annual Meeting.

Techniques: Variant Assay, Expressing, Cell Culture

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Schemes of the workflow used in this study. (A) Main steps employed in the screening along with the number of compounds left after each step. (B) A scheme showing the detailed order of utilized techniques, especially docking to CB2 structures from PDB IDs 5ZTY and 6KPC and to the CB2 model based on MD of PDB ID 6PT0 .

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques:

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: CB2–ligand complexes. (A–C) Binding sites with ligands (green) and amino acids (gray) important for ligand binding depicted in stick representation. PDB IDs 5ZTY , 6KPC , and 6PT0 , respectively. (D–F) 2D interaction schemes generated using Schrödinger Maestro. Additionally, we marked with gray, dashed circles the amino acids that are too far away from the ligand to create protein–ligand interactions in deposited structures but probably do so alternately, for limited periods of time in natural, nonstatic complexes.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Binding Assay, Ligand Binding Assay, Generated

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: (A) Radioligand displacement curves for two screened compounds with the lowest K i values toward human CB2—AS-5 and AS-7. WIN 55,212-2 was issued as the reference compound. Both identified CB2 ligands exhibit desired nanomolar K i and structural distinctiveness compared to the other known compounds with high affinity for CB2. (B) Inhibition of CP-55,940-stimulated [ 35 S]GTPγS at the CB2 receptor by the compounds at 10 μM. Results were expressed as mean percent of basal [ 35 S]GTPγS binding in the presence of 100 nM CP-55,940 as stimulating ligand. AM-630 served as a reference CB2 antagonist. Basal binding was set to 100% and is represented by the dotted line. Data was collected from three separate experiments and analyzed with the two-tailed t test. Statistical significance was depicted as follows: ** p < 0.01; *** p < 0.001.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Inhibition, Binding Assay, Two Tailed Test

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Best identified compound—AS-7 (green) docked to CB2 models based on PDB IDs 5ZTY (A), 6KPC (B) and 6PT0 MD-derived structure (C). Yellow dashed line, H-bond; teal dashed line, π–π interaction. (D) CB2–WIN 55,212-2 (magenta) complex from the largest 6PT0 MD cluster with AS-7 (green) docked to this model. The superposition shows, that despite the different chemotypes, the binding modes of both ligands exhibit similarities, mainly in the placement of the morpholine moieties and carbonyl oxygen atoms and to a lesser extent in the location of two AS-7 benzene rings in similar positions to WIN 55,212-2 central tricyclic moiety and naphthyl group.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Derivative Assay, Binding Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: AS-5 (green) docked to CB2 models based on PDB IDs 5ZTY (A) and 6PT0 MD-derived structure (B). Yellow dashed line, H-bond; teal dashed line, π–π interaction.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Derivative Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: CB2 Structures Deposited in PDB a

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Selected Results of the K i Determination with [ 3 H]CP-55,940 Displacement Assay

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Docking and MM–GBSA Results for the Four Most Potent Compounds from the In Vitro Assay and Three Already Known CB2 Ligands for Comparison

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: In Vitro, Comparison

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: HIV-infected MDM keep expressing surface CB2R at day 12pi. MDM were labeled with a fluorescent CB2R monoclonal antibody (FL4 channel) conjugated with Alexa Fluor® 647 and then analyzed by flow cytometry. ( a ) Representative histograms of extracellular staining show significant differences in fluorescence intensities from HIV + (dark histogram) and uninfected (grey histogram) on the expression of CB2R compared with the nonfluorescent MDM (unshaded histograms to the left. ( b ) A graphic representation of surface CB2R levels per donor is shown. Panels a and b are representative of three different donors (n = 3). An unstained control was included as a technical negative control and is representative of 1 donor (n = 1). ( c ) Representative images of intracellular levels of CB2R and GAPDH expression using Western Blot. Images were cropped from the same blot after stripping and reprobing with a different antibody and were acquired using different exposure times. Full-length blots are presented in Supplementary Fig. 4 ( d ) A graphic representation of intracellular CB2R levels per donor is shown. Figures c and d are representative of four different donors (n = 4).

Article Snippet: MDM were incubated with a rabbit polyclonal antibody raised against the N-terminal region of human CB2R (1:100; Cayman Chemical) in blocking buffer overnight, shaking at 4ºC.

Techniques: Infection, Expressing, Labeling, Flow Cytometry, Staining, Fluorescence, Negative Control, Western Blot, Stripping Membranes

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: CB2R agonists treatment decrease HIV-1 replication and CATB secretion from MDM. MDM were infected with HIV-1 and treated with CB2R agonists, JWH-133 and HU-308. HIV-1 p24 and CATB levels were measured from supernatants of days 3, 6, 9, and 12pi by ELISA. ( a ) Temporal HIV-1 p24 levels in supernatants of HIV-infected MDM treated with JWH-133. ( b ) Temporal CATB secretion levels in supernatants of HIV-infected MDM treated with JWH-133. Figures a and b are representative of at least four different donors (n = at least 4). ( c ) Temporal HIV-1 p24 levels in supernatants of HIV-infected MDM treated with HU-308. ( d ) Temporal CATB secretion levels in supernatants of HIV-infected MDM treated with HU-308. Figures c and d are representative of at least six different donors (n = at least 6). Graphs are presented using the mean and the ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 vs. vehicle control at its respective time-point.

Article Snippet: MDM were incubated with a rabbit polyclonal antibody raised against the N-terminal region of human CB2R (1:100; Cayman Chemical) in blocking buffer overnight, shaking at 4ºC.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: JWH-133 prevents HIV-induced increase in surface CB2R expression and induces oscillating expressions over time. MDM were cultured in 8-well chamber slides, infected with HIV-1 ADA , and treated with JWH-133 at 0.5 µM. Slides were fixed at days 3, 6, 9, and 12dpi. Cells were stained with an anti-CB2R antibody (red) and nuclei were stained with DAPI (blue). At least 3 different random pictures per condition were acquired. Representative immunofluorescence images are shown. Graphs are presented using the mean ± standard error of the mean (SEM). This figure is representative of three different donors (n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: MDM were incubated with a rabbit polyclonal antibody raised against the N-terminal region of human CB2R (1:100; Cayman Chemical) in blocking buffer overnight, shaking at 4ºC.

Techniques: Expressing, Cell Culture, Infection, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: JWH-133 decreases HIV-1 replication and CATB secretion through CB2R activation. MDM were cultured in 24-wells plates and infected with HIV-1 ADA . After removal of residual virus, cells were treated with CB2R antagonist SR144528 (SR: 1 µM) for 1 h, followed by JWH-133 (JWH) treatment at 0.5 µM. Treatments were maintained for 6dpi, exchanging half of the media at day 3pi and repeating the co-administration protocol. HIV-1 p24 and CATB levels were measured in HIV-infected MDM supernatants using ELISA. HIV-1 p24 and CATB levels were normalized against its vehicle control per donor. ( a ) HIV-1 p24 levels in HIV-infected MDM after co-administration of CB2R ligands. This figure is representative of at least eight different donors (n = 8). ( b ) CATB levels in HIV-infected MDM after co-administration of CB2R ligands. This figure is representative of ten different donors (n = 10). Graphs are presented using the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01.

Article Snippet: MDM were incubated with a rabbit polyclonal antibody raised against the N-terminal region of human CB2R (1:100; Cayman Chemical) in blocking buffer overnight, shaking at 4ºC.

Techniques: Activation Assay, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay

Journal: Biology of reproduction

Article Title: Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.

doi: 10.1095/biolreprod.103.021519

Figure Lengend Snippet: FIG. 1. Evaluation of the amounts of CB1 and CB2 during meiotic pro- gression of porcine oocytes cultured in vitro. A) Protein levels of CB1 (upper panel), CB2 (middle panel), and cdc2 (lower panel) evaluated by immunoblotting of porcine oocytes. Ten oocytes were subjected to the immunoblotting at each of the indicated culture periods. Different parts of the same membrane were used for detection of each protein. B) The amount of CB1 (upper panel), CB2 (middle panel), and cdc2 (lower panel) in immature porcine oocytes. The 200 noncultured oocytes and an in- creasing number of 24-h cultured oocytes were used for immunoblotting. Different parts of the same membrane were used for detection of each protein. C) Relative MPF activity of the porcine oocytes based on the value of 0 h. The experiments were repeated at least three times, and the values represent the means 6 standard error of the mean. D) Percentages of oocytes in which GVBD occurred (black circle), at first metaphase (black bar), at first anaphase and telophase (dotted bar), and at second metaphase (hatched bar). The numbers in parentheses represent the total number of oocytes examined.

Article Snippet: After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-CB1 monoclonal antibody (CB169, Upstate Biochemistry Inc., Waltham, MA); anti-CB2 polyclonal antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA); or anti-cdc2 monoclonal antibody [27].

Techniques: Cell Culture, In Vitro, Western Blot, Membrane, Activity Assay

Journal: Biology of reproduction

Article Title: Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.

doi: 10.1095/biolreprod.103.021519

Figure Lengend Snippet: FIG. 2. Preparation of antisense RNAs. A) Schematic representation of porcine CB1 and CB2 antisense RNAs. Positions of PCR primers are shown by small arrows and porcine antisense RNAs are shown by large arrows with length in bases. The number in parentheses represents the expected length in human CB2. B) Alignment of PCR product of porcine CB2 with the corresponding part of human CB2. Eighty-eight percent of the bases were identical and are shown by vertical bars.

Article Snippet: After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-CB1 monoclonal antibody (CB169, Upstate Biochemistry Inc., Waltham, MA); anti-CB2 polyclonal antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA); or anti-cdc2 monoclonal antibody [27].

Techniques:

Journal: Biology of reproduction

Article Title: Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.

doi: 10.1095/biolreprod.103.021519

Figure Lengend Snippet: FIG. 3. Effects of CB1 antisense RNA injection on meiotic maturation of porcine oocytes. About 25 pg/50 pl of CB1 antisense RNA was injected into porcine oocyte cytoplasm at 0 h of the culture period. A) Protein levels of CB1 (upper panel), CB2 (middle panel), and cdc2 (lower panel) of the injected porcine oocytes. Different parts of the same membrane were used for each immunoblotting. A positive control (human carcinoma cell lysate) was shown as C. B) Relative MPF activity of the injected por- cine oocytes based on the value of 0 h. The activity of noninjected oocytes shown in Figure 1B is also shown by dotted white bar for comparison. The experiments were repeated at least three times, and the values rep- resent the means 6 standard error of the mean. The bars with asterisks were significantly different from noninjected oocytes (P , 0.05). C) Per- centages of the injected oocytes in which GVBD occurred (black circle), at first metaphase (black bar), at first anaphase and telophase (dotted bar), and at second metaphase (hatched bar). The values of noninjected oocytes shown in Figure 1A are also shown by dotted line and bars for compar- ison. The numbers in parentheses represent the total number of oocytes examined. The values with asterisks were significantly different from non- injected oocytes (P , 0.05).

Article Snippet: After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-CB1 monoclonal antibody (CB169, Upstate Biochemistry Inc., Waltham, MA); anti-CB2 polyclonal antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA); or anti-cdc2 monoclonal antibody [27].

Techniques: Injection, Membrane, Western Blot, Positive Control, Activity Assay, Comparison

Journal: Biology of reproduction

Article Title: Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.

doi: 10.1095/biolreprod.103.021519

Figure Lengend Snippet: FIG. 4. Effects of CB2 antisense RNA injection on meiotic maturation of porcine oocytes. About 25 pg/50 pl of CB2 antisense RNA was injected into porcine oocyte cytoplasm at 0 h of the culture period. For a detailed explanation, see the legend of Figure 3.

Article Snippet: After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-CB1 monoclonal antibody (CB169, Upstate Biochemistry Inc., Waltham, MA); anti-CB2 polyclonal antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA); or anti-cdc2 monoclonal antibody [27].

Techniques: Injection

Journal: Biology of reproduction

Article Title: Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.

doi: 10.1095/biolreprod.103.021519

Figure Lengend Snippet: FIG. 5. Effects of CB1 and CB2 antisense RNA mixture injection on mei- otic maturation of porcine oocytes. About 50 pl mixture of CB1 antisense RNA (25 pg) and CB2 antisense RNA (25 pg) was injected into porcine oocyte cytoplasm at 0 h of the culture period. For a detail explanation, see the legend of Figure 3. No injected oocyte was at second metaphase at 48 h of culture.

Article Snippet: After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-CB1 monoclonal antibody (CB169, Upstate Biochemistry Inc., Waltham, MA); anti-CB2 polyclonal antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA); or anti-cdc2 monoclonal antibody [27].

Techniques: Injection

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cannabinoid CB 1 Receptor Is Downregulated in Clear Cell Renal Cell Carcinoma

doi: 10.1369/jhc.2010.957126

Figure Lengend Snippet: mRNA expression of CB1 receptors in kidney tumor (T) and surrounding normal tissue (N). Agarose gel showing the presence of a single transcript, corresponding to the expected product size encoding the cannabinoid CB1 receptor (131 bp, CNR1). The mRNA encoding the 110-bp product expected for the cannabinoid CB2 receptor (CNR2) was undetectable. The expression of SHDA, used as an internal control, is also shown. M, molecular size standards; +, positive control showing the expression of all genes in a pool of cDNAs from 20 different human tissues; −, negative control with no RNA in the reverse transcriptase reaction.

Article Snippet: Blots were blocked in milk and incubated overnight with the CB 1 (1:250 dilution; catalog ref. PA1-743, ABR Affinity BioReagents, Golden, CO) and CB 2 (1:200 dilution; catalog ref. 101550, Cayman Chemical, Ann Arbor, MI) polyclonal antibodies.

Techniques: Expressing, Agarose Gel Electrophoresis, Positive Control, Negative Control