Journal: Journal of Inflammation (London, England)

Article Title: Cannabinoid 2 receptor attenuates inflammation during skin wound healing by inhibiting M1 macrophages rather than activating M2 macrophages

doi: 10.1186/s12950-018-0201-z

Figure Lengend Snippet: a and b Representative immunoblotting results and ( c ) semi-quantitative analysis of CB2R protein level at different posttraumatic intervals. + P < 0.05, ++ P < 0.01, JWH133 versus vehicle; * P < 0.05, ** P < 0.01, GP1a versus vehicle; # P < 0.05, ## P < 0.01, AM630 versus vehicle

Article Snippet: After being blocked with 5% fat-free milk at RT for 2 h, PVDF membranes were incubated, respectively, with a rabbit anti-CB2R polyclonal primary antibody (1:250 dilution; sc-25,494, Santa Cruz Biotechnology, CA, USA) and mouse anti–GAPDH monoclonal antibody (1:2500 dilution, sc-32,233; Santa Cruz Biotechnology, Inc) at 4 °C overnight.

Techniques: Western Blot

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: Primers used for quantification of mRNA expression in brain by RT-qPCR.

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Expressing, Sequencing

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) CB1R and (B.) CB2R mRNA were assessed in brain tissue by RT-qPCR and normalized to 18S expression. Data are expressed as fold change with respect to sham and revealed a selective 15-fold increase in CB2R expression. (C.) Histological analysis revealed increased CB2R+ immunoreactivity in the injured cortex (Scale-50µm), as compared to sham-operated brains. (D.) CB2R+ cells were quantified as mean fluorescent intensity and normalized to sham. (E.) Bone marrow chimera mice were generated whereby myeloid cells expressed red fluorescent protein (RFP). CB2R was expressed in 52.7% of RFP+CD11b+CD45HI infiltrating myeloid cells in the brain after TBI whereas 17.5% of RFP+ cells expressed CB2R in sham brains, as assessed by flow cytometry. (F) Quantification of RFP+CD11b+CD45HICB2R+ shown in panel (E.). Groups were compared as Student’s t-test. Data were represented as mean ± SEM (n=6; **p<0.01; ***p<0.001 vs. sham).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Quantitative RT-PCR, Expressing, Generated, Flow Cytometry

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) Schematic of macrophage (MΦ) adoptive transfer in recipient mice. CFSE-labeled CD11b+, CD68+, F4/80+ macrophages were intravenously administered through the tail vein immediately after sham/TBI. Brains were harvested for immunofluorescent staining at 72h post-injury. (B.) The increased infiltration of CFSE+ macrophages temporally and spatially paralleled increased CB2R+ expression after TBI (Scale-50µm). (C.) CB2R+ (red) and CFSE-labeled macrophages (green) were quantified as mean fluorescent intensity. Data were represented as mean ± SEM (n=6) and analyzed using Student’s t-test (***p<0.001 vs. sham).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Adoptive Transfer Assay, Labeling, Staining, Expressing

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) Representative flow cytometry dot plots showing CD68+ CD11b+ CB2R+ myeloid cells were sorted after sham/TBI and analyzed for microglia (CD45Low) and infiltrating macrophages (CD45High) populations. A TBI-induced increase in infiltrating macrophages was significantly reduced by treatment with GP1a (1 and 3 mg/kg), but not after treatment with the CB2R antagonist, AM360 (5 mg/kg). Interestingly, microglia did not show any significant alterations in numbers at day 3 post sham/TBI with or without treatment. Gating strategy has been shown in Fig 3A. Quantification of (B.) microglia and (C.) infiltrating macrophages are shown here. Data were represented as mean ± SEM and groups were compared by One-way ANOVA followed by Newman-Keuls multiple comparison. (n=6–7/group; *p<0.05; **p<0.01).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Flow Cytometry

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) Representative flow cytometry dot plots showing CB2R+ CD11b+ myeloid cells were sorted after sham/TBI and analyzed for M1 (CD68+ CD206− TNFα+) and M2 (CD68+ CD206+ IL10+) polarization. A TBI-induced increase in M1 polarization was significantly reduced by treatment with 3 mg/kg GP1a, but not after treatment with the CB2R antagonist, AM360 (5 mg/kg). Dot plots for GP1a treatments (1 mg/kg b.wt and 5 mg/kg b.wt.) are not shown here. Quantification of (B.) M2 macrophages (C.) M1 macrophages, and (D.) the ratio of M2:M1 macrophages after treatment. Data were represented as mean ± SEM and groups were compared by One-way ANOVA followed by Newman-Keuls multiple comparison. (n=6/group; *p<0.05; **p<0.01; ***p<0.001).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Flow Cytometry

Journal: Journal of neurochemistry

Article Title: Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

doi: 10.1111/j.1471-4159.2011.07391.x

Figure Lengend Snippet: Proteins differentially regulated by chronic Δ9-THC in intact and ovarectomized rats DeCyder software (GE Healthcare) was used for simultaneous comparison of abundance changes across all samples’ 2-DE gels, and for comparisons of individual Cy3 and Cy5 samples for each rat. The DeCyder differential in-gel analysis (DIA) module was used for pair-wise comparisons of each sample on a gel to the Cy2 labeled standard present on each gel. For each pair-wise DIA comparison, the entire signals from each CyDye channel are normalized prior to the co-detection of protein spot boundaries and the calculation of the volume ratio for each protein spot. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all protein spot maps from all gels, and using the Cy3/Cy5:Cy2 DIA ratios, calculate abundance changes and paired Student’s t -test p -values for the variance of these ratios for each protein pair across all samples. In the absence of sufficient experimental replicates, no statistical information can be generated. Fold abundance changes are reported, whereby a fold increase is calculated directly from the volume ratio, and a fold decrease by the inverse of volume ratio. The DIA module was also used to calculate the direct Cy5:Cy3 volume ratio for each paired sample individually (without the Cy2 mixed standard) to assess the contribution from each subject and reveal changes that were present in a group. After determining spots of interest, the protein were excised using the Ettan Spot Handling Workstation with a 2-mm diameter spot-picking head (GE Healthcare). Gel spots were cut, de-stained and then digested with trypsin (Promega). The resulting peptide mixture was loaded on a Dionex PepMap C18 trap column and was separated by a New Objective reversed phase C18 Picofrit column/emitter. Peptide mass was determined by a Thermo-Fisher LTQXL linear ion trap mass spectrometer (Waltham, MA, USA) coupled with an Eksigent nanoLC (Dublin, CA, USA). The raw data were analyzed by the Mascot search engine V2.2 (Matrix Science Inc, Boston, MA, USA) against the rat SwissProt database (false discovery rate <5%) to generate a list of possible proteins for that gel spot.

Article Snippet: The sources of the antibodies used in the present study were as follows: anti-CB1R and anti-CB2R antibodies were from Cayman, anti-hemagglutinin (anti-HA) was from Santa Cruz, anti-AHA1 antibody was from Abnova, anti-HSP90α and anti-HSP90β antibodies were from Enzo Lifesciences, secondary anti-mouse and anti-rabbit antibodies were from Perkin Elmer, Alexa Fluor 594-labeled anti-mouse and anti-rabbit and anti-mouse antibodies and 4,6-diamidino-2-phenylindole were obtained from Invitrogen.

Techniques: Software, Comparison, Labeling, Generated, Mass Spectrometry, Clinical Proteomics, Membrane

Journal: Journal of neurochemistry

Article Title: Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

doi: 10.1111/j.1471-4159.2011.07391.x

Figure Lengend Snippet: A. Western blots of AHA1 levels in the cerebellar extracts from intact or ovariectimized female rats administered either saline or Δ9-THC during adolescence. The cerebellar extracts (20 μg/lane) were separated by 10% SDS-PAGE, transferred onto PDVF membranes, and subjected to western-blotting with specific antibodies. The data were quantified using a Fuji Film luminescent image analyzer (LAS-1000 Plus) and the Image Gauge program (Version 3.4). B. Quantification for the experiments shown in A. AHA1 levels were corrected for the β-actin levels in the same samples and the data are presented as the % from the levels found in the control group. *- indicate statistical significant differences between the four groups with p < 0.05.

Article Snippet: The sources of the antibodies used in the present study were as follows: anti-CB1R and anti-CB2R antibodies were from Cayman, anti-hemagglutinin (anti-HA) was from Santa Cruz, anti-AHA1 antibody was from Abnova, anti-HSP90α and anti-HSP90β antibodies were from Enzo Lifesciences, secondary anti-mouse and anti-rabbit antibodies were from Perkin Elmer, Alexa Fluor 594-labeled anti-mouse and anti-rabbit and anti-mouse antibodies and 4,6-diamidino-2-phenylindole were obtained from Invitrogen.

Techniques: Western Blot, Saline, SDS Page, Control

Journal: Journal of neurochemistry

Article Title: Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

doi: 10.1111/j.1471-4159.2011.07391.x

Figure Lengend Snippet: A. AHA-1 levels in control and AHA-1 transfected HEK293T cells. The cells were transfected with pcDNA 3.1 or AHA-1 (2.25 μg/well in 6-well plates) and serum starved for 24 h. Two days after transfection the cells were lysated and 10 μg of total protein were separated by 10% SDS-PAGE and AHA-1 levels were determined by western blot. B. Modulation of CB1R and CB2R plasma membrane levels by AHA1 overexpression. HEK293T were co-transfected with CB1R or CB2R (0.25 μg/well) and pcDNA 3.1 or AHA-1 (2.25 μg/well each) and after six hours the cells were trypsinized and plated on 12-well plates as described in Material and Methods. FBS was withdrawn for 24 h, and two days after transfection the plasma membrane levels of CB1R and CB2R were determined by ELISA as described in Material and Methods. n=12 in each case from three different transfections. *- indicate p < 0.05 compared to pcDNA 3.1 transfected cells. C. Subcellular localization of CB1R in HEK293T cells. The cells were grown on coverslips in 6-well plates and transfected with 3xHA-CB1R (0.1 μg/well) and DsRed-Rab5 (0.1 μg/well). After serum starvation for 24 h, the cells were fixed and permeabilized. The CB1R localization was stained using HA antibody as described in Material and Methods. Blue: DNA staining by 4,6-diamidino-2-phenylindole (nuclear), green: 3xHA-CB1R, red: DsRed-Rab5. The images are representative from three different coverslips, obtained from three independent transfections. D. CB1R interacts with AHA-1 in HEK293T cells. The cells were transiently co-transfected in 10 mm2 dishes with pcDNA 3.1 (10 μg, control), or with CB1R (5 μg) and pcDNA3.1 (5 μg), or CB1R (5 μg) and AHA-1 (5 μg). After two days the cells were solubilized and immunoprecipitated with CB1R antibody as described under Material and Methods. The immunoprecipitates (20 μg/lane) or the lysates (10 μg/lane) were separated by 10 % SDS-Page and subject to western-blotting with AHA-1 and CB1R antibodies. The experiment shown is representative from three independent transfections.

Article Snippet: The sources of the antibodies used in the present study were as follows: anti-CB1R and anti-CB2R antibodies were from Cayman, anti-hemagglutinin (anti-HA) was from Santa Cruz, anti-AHA1 antibody was from Abnova, anti-HSP90α and anti-HSP90β antibodies were from Enzo Lifesciences, secondary anti-mouse and anti-rabbit antibodies were from Perkin Elmer, Alexa Fluor 594-labeled anti-mouse and anti-rabbit and anti-mouse antibodies and 4,6-diamidino-2-phenylindole were obtained from Invitrogen.

Techniques: Control, Transfection, SDS Page, Western Blot, Clinical Proteomics, Membrane, Over Expression, Enzyme-linked Immunosorbent Assay, Staining, Immunoprecipitation

Journal: Journal of neurochemistry

Article Title: Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

doi: 10.1111/j.1471-4159.2011.07391.x

Figure Lengend Snippet: HEK293T co-transfected with CB1R or CB2R and pcDNA3.1 or AHA1 as in Fig 2B were plated on 24 well-plates and serum straved for 24h. Two days after transfection the medium was changed to PBS containing 100 μM IBMX for one hour. Subsequently, the cells were pretreated with Δ9-THC (1 μM)) for 5 min, followed by stimulation with forskolin (10 μM) for 15 min. The reactions were stopped by medium aspiration and addition of 200 μl thhricloracetic acid. cAMP levels were determined using cAMP Elisa Kit (Cayman Biochemicals) as described in Material and Methods. n=12 in each case from three independent transfections. *- indicates statistically significant differences between pcDNA 3.1 and AHA1 transfected cells by two-way ANOVA followed by Holm Sidak-test (interaction: F(2,12=14.03, p<0.001).

Article Snippet: The sources of the antibodies used in the present study were as follows: anti-CB1R and anti-CB2R antibodies were from Cayman, anti-hemagglutinin (anti-HA) was from Santa Cruz, anti-AHA1 antibody was from Abnova, anti-HSP90α and anti-HSP90β antibodies were from Enzo Lifesciences, secondary anti-mouse and anti-rabbit antibodies were from Perkin Elmer, Alexa Fluor 594-labeled anti-mouse and anti-rabbit and anti-mouse antibodies and 4,6-diamidino-2-phenylindole were obtained from Invitrogen.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay

Journal: Journal of neurochemistry

Article Title: Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

doi: 10.1111/j.1471-4159.2011.07391.x

Figure Lengend Snippet: HEK293T cells in 6-well plates were co-transfected with CB1R (0.25/μg/well) and pcDNA 3.1 or AHA1 (each at 2.25 μg/well). Subsequently, the cells were serum starved for 24 h and then stimulated with increasing concentrations of Δ9-THC for 5 min. The reactions were stopped by aspiration of the medium and addition of 200 μl lysis buffer. Ten μg of protein were separated by SDS-page and ERK 1/2 activation and total ERK 1/2 levels were detected using specific antibodies. Top panel: representative blots from four different experiments. Lower panel: Quantification of the effects of Δ9-THC on MAPK levels, open symbols indicate pcDNA 3.1 transfected cells and black symbols indicate AHA1 overexpressing cells; n=4 in each case from four different transfections, * indicates statistically significant differences compared to pcDNA 3.1 transfected cells by one-way ANOVA.

Article Snippet: The sources of the antibodies used in the present study were as follows: anti-CB1R and anti-CB2R antibodies were from Cayman, anti-hemagglutinin (anti-HA) was from Santa Cruz, anti-AHA1 antibody was from Abnova, anti-HSP90α and anti-HSP90β antibodies were from Enzo Lifesciences, secondary anti-mouse and anti-rabbit antibodies were from Perkin Elmer, Alexa Fluor 594-labeled anti-mouse and anti-rabbit and anti-mouse antibodies and 4,6-diamidino-2-phenylindole were obtained from Invitrogen.

Techniques: Transfection, Lysis, SDS Page, Activation Assay

Journal: Journal of neurochemistry

Article Title: Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

doi: 10.1111/j.1471-4159.2011.07391.x

Figure Lengend Snippet: A. AHA-1 levels in control and AHA-1 transfected Neuro-2A cells. The cells were transfected with pcDNA 3.1 or AHA-1 (2.25 μg/well in 6-well plates) and serum starved for 24 h. The lysates and the western blot were performed as described in Fig 2A. B. Cell surface levels of CB1R in Neuro-2A overexpressing cells. Neuro2-A cells were transfected as above and then trypsinized and plated at a density of 50×104 cells/well in 12-well plates. Subsequent procedures to determine plasma membrane CB1R levels were similar to that described for HEK293T cells in Fig 2B. n=9 from three different experiments. * indicates statistically significant differences compared to pcDNA 3.1 transfected cells. C. The effects of CB1R stimulation on cAMP levels in pcDNA 3.1 or AHA-1 transfected Neuro-2A cells. The transfections were made as described and subsequently, the cAMP determinations were conducted as described in the Fig 3. n=12 in each case from three different transfections; * - indicates statistically significant differences between pcDNA 3.1 and AHA1 transfected cells by two-way ANOVA followed by Holm Sidak-test (interaction: F(2,12=30.98, p<0.001).

Article Snippet: The sources of the antibodies used in the present study were as follows: anti-CB1R and anti-CB2R antibodies were from Cayman, anti-hemagglutinin (anti-HA) was from Santa Cruz, anti-AHA1 antibody was from Abnova, anti-HSP90α and anti-HSP90β antibodies were from Enzo Lifesciences, secondary anti-mouse and anti-rabbit antibodies were from Perkin Elmer, Alexa Fluor 594-labeled anti-mouse and anti-rabbit and anti-mouse antibodies and 4,6-diamidino-2-phenylindole were obtained from Invitrogen.

Techniques: Control, Transfection, Western Blot, Clinical Proteomics, Membrane

Journal: Journal of Biological Chemistry

Article Title: The Endocannabinoid System in Human Keratinocytes

doi: 10.1074/jbc.m303994200

Figure Lengend Snippet: FIG. 3. Changes of the endocannabinoid system in differentiating HaCaT cells. A, time dependence of cannabinoid receptor (CBR) binding, AEA membrane transporter (AMT) activity, AEA hydrolase (FAAH) activity, and AEA-synthesizing phospholipase D (PLD) activity in HaCaT cells induced to differentiate by TPA plus calcium (see “Experimental Procedures” for details). 100% 370 36 fmolmg protein1 for CBR binding (substrate: 200 pM [3H]CP55.940), 70 6 pmolmin1mg protein1 for AMT (substrate: 400 nM [3H]AEA), 105 10 pmolmin1mg protein1 for FAAH (substrate: 5 M [3H]AEA), and 110 10 pmolmin1mg protein1 for PLD (substrate: 10 M 1,2-dioleoyl-3-phosphatidyl-[2- 14C]ethanolamine). Vertical bars represent S.D. values. *, p 0.05; **, p 0.01 versus controls (p 0.05 in all other cases). B, Western blot analysis of FAAH expression in differentiating HaCaT cell extracts, reacted with anti-FAAH polyclonal (upper panel) or anti-actin monoclonal (lower panel) antibodies. Molecular mass markers are shown on the right. C, RT-PCR analysis of cDNA of the same samples as in B. The expected sizes of the amplicons (199 bp for FAAH and 258 bp for 18 S rRNA) are shown on the right.

Article Snippet: Rabbit anti-CB1R and anti-CB2R polyclonal antibodies were from Cayman Chemicals, and mouse anti-actin monoclonal antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Binding Assay, Membrane, Activity Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction