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Santa Cruz Biotechnology short hairpin rna shrna targeting cb2
Short Hairpin Rna Shrna Targeting Cb2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(A) Reduced CB2 mRNA levels in cells treated with CB2 shRNA lentiviral particles. (**p<0.01 significant effect of CB2 shRNA compared to control shRNA treated cells). (B) Reduced CB2 protein levels in cells treated with CB2 shRNA lentiviral particles compared to control shRNA lentiviral particle treated controls. (C) CB2 shRNA lentivirus transfection prevents CP55,940, JWH133 and GP1a-induced increases in 5-HT2A receptor mRNA. **p<0.01, significant effect of CP55,940, JWH133, and GP1a treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus transfected cells compared to vehicle-treated controls. ##p<0.01, significant effect of CB2 shRNA lentivirus transfection on the CP55,940, JWH133, and GP1a-induced upregulation of 5-HT2A receptors. The data represent mean ± SEM (n=3).

Journal: European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology

Article Title: Cannabinoid 2 Receptor- and Beta Arrestin 2-Dependent Upregulation of Serotonin 2A Receptors

doi: 10.1016/j.euroneuro.2012.06.012

Figure Lengend Snippet: (A) Reduced CB2 mRNA levels in cells treated with CB2 shRNA lentiviral particles. (**p<0.01 significant effect of CB2 shRNA compared to control shRNA treated cells). (B) Reduced CB2 protein levels in cells treated with CB2 shRNA lentiviral particles compared to control shRNA lentiviral particle treated controls. (C) CB2 shRNA lentivirus transfection prevents CP55,940, JWH133 and GP1a-induced increases in 5-HT2A receptor mRNA. **p<0.01, significant effect of CP55,940, JWH133, and GP1a treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus transfected cells compared to vehicle-treated controls. ##p<0.01, significant effect of CB2 shRNA lentivirus transfection on the CP55,940, JWH133, and GP1a-induced upregulation of 5-HT2A receptors. The data represent mean ± SEM (n=3).

Article Snippet: Lentivirus and stable transduction of shRNAs in CLU213 cells β-Arrestin 2 shRNA (r), CB2 shRNA (r), copGFP control, control shRNA lentiviral particles, polybrene, and puromyocin were purchased from Santa Cruz, CA.

Techniques: shRNA, Control, Transfection

(A) Reduced β-Arr2 mRNA levels in cells treated with β-Arr2 shRNA lentiviral particles. (**p<0.01 significant effect of β-Arr2 shRNA compared to control shRNA treated cells). (B) Reduced β-Arr2 protein levels in cells treated with β-Arr2 shRNA lentiviral particles compared to control shRNA lentiviral particle treated controls. (C) β-Arr2 shRNA lentivirus transfection prevents CP55,940, JWH133 and GP1a-induced increases in 5-HT2A receptor mRNA. **p<0.01, significant effect of CP55,940, JWH133, and treatment on 5-HT2A receptor mRNA levels in control shRNA transfected cells compared to vehicle-treated controls. ##p<0.01 or #p<0.05, significant effect of β-Arr2 shRNA transfection on the CP55,940 and JWH133 or GP1a-induced upregulation of 5-HT2A receptors. @p<0.05, significant effect of GP 1a treatment in β-Arr2 shRNA transfected cells compared to vehicle-treated β-Arr2 shRNA transfected cells. The data represent mean ± SEM (n=3).

Journal: European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology

Article Title: Cannabinoid 2 Receptor- and Beta Arrestin 2-Dependent Upregulation of Serotonin 2A Receptors

doi: 10.1016/j.euroneuro.2012.06.012

Figure Lengend Snippet: (A) Reduced β-Arr2 mRNA levels in cells treated with β-Arr2 shRNA lentiviral particles. (**p<0.01 significant effect of β-Arr2 shRNA compared to control shRNA treated cells). (B) Reduced β-Arr2 protein levels in cells treated with β-Arr2 shRNA lentiviral particles compared to control shRNA lentiviral particle treated controls. (C) β-Arr2 shRNA lentivirus transfection prevents CP55,940, JWH133 and GP1a-induced increases in 5-HT2A receptor mRNA. **p<0.01, significant effect of CP55,940, JWH133, and treatment on 5-HT2A receptor mRNA levels in control shRNA transfected cells compared to vehicle-treated controls. ##p<0.01 or #p<0.05, significant effect of β-Arr2 shRNA transfection on the CP55,940 and JWH133 or GP1a-induced upregulation of 5-HT2A receptors. @p<0.05, significant effect of GP 1a treatment in β-Arr2 shRNA transfected cells compared to vehicle-treated β-Arr2 shRNA transfected cells. The data represent mean ± SEM (n=3).

Techniques: shRNA, Control, Transfection

(A) ConA pretreatment prevents CP55,940 and GP1a-induced increases in 5-HT2A receptor mRNA. **p<0.01, significant effect of CP55,940 and GP1a treatment on 5-HT2A receptor mRNA levels compared to vehicle-treated controls. ##p<0.01, significant effect of ConA pretreatment on the CP55,940 and GP1a-induced upregulation of 5-HT2A receptors. (B) ConA pretreatment prevents CP55,940 and GP1a-induced increases in cytosolic-associated CB2 receptor protein levels. **p<0.01, significant effect of CP55,940 and GP1a treatment on cytosolic CB2 receptor protein levels compared to vehicle-treated controls. ##p<0.01, significant effect of ConA pretreatment on the CP55,940 and GP1a-induced increases in cytosolic CB2 receptor protein levels. @p<0.05, significant effect CP 55,940 treatment in ConA pretreated cells compared to ConA/vehicle or ConA/GP1a treated cells. (C) JWH-073 does not significantly (p>0.05) alter 5-HT2A receptor mRNA levels. **p<0.01, significant effect of CP55,940 and GP1a treatment on 5-HT2A receptor mRNA levels compared to vehicle-treated controls. (D) ConA pretreatment prevents GP1a-induced increases in nuclear pERK. **p<0.01, significant effect of GP1a treatment on nuclear pERK levels compared to vehicle-treated controls. ##p<0.01, significant effect of ConA pretreatment on GP1a-induced increases in nuclear pERK levels. The data represent mean ± SEM (n=3).

Journal: European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology

Article Title: Cannabinoid 2 Receptor- and Beta Arrestin 2-Dependent Upregulation of Serotonin 2A Receptors

doi: 10.1016/j.euroneuro.2012.06.012

Figure Lengend Snippet: (A) ConA pretreatment prevents CP55,940 and GP1a-induced increases in 5-HT2A receptor mRNA. **p<0.01, significant effect of CP55,940 and GP1a treatment on 5-HT2A receptor mRNA levels compared to vehicle-treated controls. ##p<0.01, significant effect of ConA pretreatment on the CP55,940 and GP1a-induced upregulation of 5-HT2A receptors. (B) ConA pretreatment prevents CP55,940 and GP1a-induced increases in cytosolic-associated CB2 receptor protein levels. **p<0.01, significant effect of CP55,940 and GP1a treatment on cytosolic CB2 receptor protein levels compared to vehicle-treated controls. ##p<0.01, significant effect of ConA pretreatment on the CP55,940 and GP1a-induced increases in cytosolic CB2 receptor protein levels. @p<0.05, significant effect CP 55,940 treatment in ConA pretreated cells compared to ConA/vehicle or ConA/GP1a treated cells. (C) JWH-073 does not significantly (p>0.05) alter 5-HT2A receptor mRNA levels. **p<0.01, significant effect of CP55,940 and GP1a treatment on 5-HT2A receptor mRNA levels compared to vehicle-treated controls. (D) ConA pretreatment prevents GP1a-induced increases in nuclear pERK. **p<0.01, significant effect of GP1a treatment on nuclear pERK levels compared to vehicle-treated controls. ##p<0.01, significant effect of ConA pretreatment on GP1a-induced increases in nuclear pERK levels. The data represent mean ± SEM (n=3).

Techniques:

FIGURE 1. CP55940-induced enhanced phosphorylation of CB2 receptors, increased GRK5 expression levels, and reduced GRK2 expression levels in rat PFCx. Rats were injected with CP55940 (0.05 mg/kg, intraperitoneally) once a day for 7 days. After decapitation, the brains were collected, and PFCx was dissected. A, phosphorylated proteins were separated and detected as described under “Experimental Procedures.” 30 g of isolated phosphorylated protein was used in Western blot detection. B–E, CB2 receptor and GRK protein levels were evaluated by Western blot. Proteins (8 g) were resolved by SDS-PAGE, and antibodiesforCB2receptor(AandB),GRK5(C),GRK6(D),andGRK2(E)wereusedtodetecttheproteinsofinterest.RepresentativeWesternblotsareshown,and integratedopticaldensitywascalculatedasdescribedunder“ExperimentalProcedures.”-Actinwasusedasaloadingcontrol.F,GRK5,GRK6,andGRK2mRNA levels were evaluated by qRT-PCR as described under “Experimental Procedures.” **, p 0.01; *, p 0.05, significant effect of CP55940 treatment compared with vehicle-treated controls. The data represent mean S.E. (error bars) (n 6–8).

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 1. CP55940-induced enhanced phosphorylation of CB2 receptors, increased GRK5 expression levels, and reduced GRK2 expression levels in rat PFCx. Rats were injected with CP55940 (0.05 mg/kg, intraperitoneally) once a day for 7 days. After decapitation, the brains were collected, and PFCx was dissected. A, phosphorylated proteins were separated and detected as described under “Experimental Procedures.” 30 g of isolated phosphorylated protein was used in Western blot detection. B–E, CB2 receptor and GRK protein levels were evaluated by Western blot. Proteins (8 g) were resolved by SDS-PAGE, and antibodiesforCB2receptor(AandB),GRK5(C),GRK6(D),andGRK2(E)wereusedtodetecttheproteinsofinterest.RepresentativeWesternblotsareshown,and integratedopticaldensitywascalculatedasdescribedunder“ExperimentalProcedures.”-Actinwasusedasaloadingcontrol.F,GRK5,GRK6,andGRK2mRNA levels were evaluated by qRT-PCR as described under “Experimental Procedures.” **, p 0.01; *, p 0.05, significant effect of CP55940 treatment compared with vehicle-treated controls. The data represent mean S.E. (error bars) (n 6–8).

Article Snippet: MAY 31, 2013 • VOLUME 288 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15713 by guest on June 28, 2015 http://w w w .jbc.org/ D ow nloaded from Lentivirus and Stable Transduction of shRNAs in CLU213 Cells—GRK5 shRNA (rat), -arrestin 2 shRNA (rat), CB1 shRNA (rat), CB2 shRNA (rat), control shRNA lentiviral particles, Polybrene, and puromyocin were purchased from Santa Cruz Biotechnology, Inc. Optimal transduction conditions were determined, and transfection of cells with lentiviral particles was conducted as described previously (3).

Techniques: Phospho-proteomics, Expressing, Injection, Isolation, Western Blot, SDS Page, Quantitative RT-PCR

FIGURE 3. GP1a, a selective CB2 receptor agonist, up-regulated GRK5 via CB2 receptors, -arrestin 2, and ERK1/2 signaling in CLU213 cells. A, cells were incubated with either vehicle (ethanol 0.01% final concentration), 1 nM GP1a, or 15 nM ACEA for 72 h. qRT-PCR was used to show the effect of selective CB1 or CB2 receptor agonist treatment on GRK5 mRNA levels. **, p 0.01 significant effect of GP1a treatment compared with vehicle-treated controls. B, cells stably transfected with control, CB1, or CB2 shRNA lentiviral particles were treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to examine the effect of CB1 or CB2 receptor knockdown on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment on GRK5 mRNA levels in control or CB1 shRNA lentivirus-treated cells compared with vehicle-treated controls. ##, p 0.01, significant effect of CB2 shRNA lentivirus transfection on the GP1a-induced up-regulation of GRK5. *, p 0.05, significant effect of GP1a treatment in CB2 shRNA-transfected cells compared with vehicle-treated/CB2 shRNA-transfected cells. C, cells were pretreated with vehicle or 200 nM PD198306, potent ERK1/2 inhibitor, and then treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to show the effect of PD198306 pretreatment on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment compared with vehicle-treated controls. ##, p 0.01, significant effect of PD198306 pretreatment on GP1a-induced increases in GRK5 mRNA levels compared with vehicle-treated controls. D, cells were stably transfected with control or -arrestin 2 shRNA lentivirus particles and then treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to examine the effect of -arrestin 2 knockdown on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment on GRK5 mRNA levels in control shRNA lentivirus-treated cells compared with vehicle-treated controls. ##, p 0.01, significant effect of -arrestin 2 shRNA lentivirus transfection on the GP1a-induced up-regulation of GRK5. *, p 0.05, significant effect of GP1a treatment in -arrestin 2 shRNA-transfected cells compared with vehicle-treated -arrestin 2 shRNA-transfected cells. The data represent mean S.E. (error bars) (n 3).

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 3. GP1a, a selective CB2 receptor agonist, up-regulated GRK5 via CB2 receptors, -arrestin 2, and ERK1/2 signaling in CLU213 cells. A, cells were incubated with either vehicle (ethanol 0.01% final concentration), 1 nM GP1a, or 15 nM ACEA for 72 h. qRT-PCR was used to show the effect of selective CB1 or CB2 receptor agonist treatment on GRK5 mRNA levels. **, p 0.01 significant effect of GP1a treatment compared with vehicle-treated controls. B, cells stably transfected with control, CB1, or CB2 shRNA lentiviral particles were treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to examine the effect of CB1 or CB2 receptor knockdown on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment on GRK5 mRNA levels in control or CB1 shRNA lentivirus-treated cells compared with vehicle-treated controls. ##, p 0.01, significant effect of CB2 shRNA lentivirus transfection on the GP1a-induced up-regulation of GRK5. *, p 0.05, significant effect of GP1a treatment in CB2 shRNA-transfected cells compared with vehicle-treated/CB2 shRNA-transfected cells. C, cells were pretreated with vehicle or 200 nM PD198306, potent ERK1/2 inhibitor, and then treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to show the effect of PD198306 pretreatment on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment compared with vehicle-treated controls. ##, p 0.01, significant effect of PD198306 pretreatment on GP1a-induced increases in GRK5 mRNA levels compared with vehicle-treated controls. D, cells were stably transfected with control or -arrestin 2 shRNA lentivirus particles and then treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to examine the effect of -arrestin 2 knockdown on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment on GRK5 mRNA levels in control shRNA lentivirus-treated cells compared with vehicle-treated controls. ##, p 0.01, significant effect of -arrestin 2 shRNA lentivirus transfection on the GP1a-induced up-regulation of GRK5. *, p 0.05, significant effect of GP1a treatment in -arrestin 2 shRNA-transfected cells compared with vehicle-treated -arrestin 2 shRNA-transfected cells. The data represent mean S.E. (error bars) (n 3).

Techniques: Incubation, Concentration Assay, Quantitative RT-PCR, Stable Transfection, Transfection, Control, shRNA, Knockdown

FIGURE 4. GRK5 is necessary for the CP55940 and GP1a-induced up-reg- ulation of 5-HT2A receptors in CLU213 cells. Cells were transfected with control or GRK5 shRNA lentiviral particles as described under “Experimental Procedures.” A, qRT-PCR to show reduced GRK5 mRNA levels in cells treated with GRK5 shRNA lentiviral particles. B, Western blot to show reduced GRK5 protein levels in cells treated with GRK5 shRNA lentiviral particles. A and B, **, p 0.01, significant effect of GRK5 shRNA compared with control shRNA- treated cells. C and D, cells stably transfected with control or GRK5 shRNA lentiviral particles were treated with either vehicle, 1 nM CP55940, or 1 nM GP1a for 72 h. C, qRT-PCR to show the effect of GRK5 knockdown on CP55940- induced increases on 5-HT2A receptor mRNA levels. **, p 0.01, significant effect of CP55940 treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lentivirus transfection on the CP55940-induced up-regulation of 5-HT2A receptors. **, p 0.01, significant effect of CP55940 treatment in GRK5 shRNA-transfected cells compared with vehicle-treated GRK5 shRNA-transfected cells. D, qRT-PCR to show the effect of GRK5 knockdown on GP1a-induced increases in 5-HT2A receptor mRNA levels. **, p 0.01, significant effect of GP1a treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lenti- virustransfectionontheGP1a-inducedup-regulationof5-HT2Areceptors.**, p 0.01, significant effect of GP1a treatment in GRK5 shRNA-transfected cells compared with vehicle-treated GRK5 shRNA-transfected cells. The data rep- resent mean S.E. (error bars) (n 3).

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 4. GRK5 is necessary for the CP55940 and GP1a-induced up-reg- ulation of 5-HT2A receptors in CLU213 cells. Cells were transfected with control or GRK5 shRNA lentiviral particles as described under “Experimental Procedures.” A, qRT-PCR to show reduced GRK5 mRNA levels in cells treated with GRK5 shRNA lentiviral particles. B, Western blot to show reduced GRK5 protein levels in cells treated with GRK5 shRNA lentiviral particles. A and B, **, p 0.01, significant effect of GRK5 shRNA compared with control shRNA- treated cells. C and D, cells stably transfected with control or GRK5 shRNA lentiviral particles were treated with either vehicle, 1 nM CP55940, or 1 nM GP1a for 72 h. C, qRT-PCR to show the effect of GRK5 knockdown on CP55940- induced increases on 5-HT2A receptor mRNA levels. **, p 0.01, significant effect of CP55940 treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lentivirus transfection on the CP55940-induced up-regulation of 5-HT2A receptors. **, p 0.01, significant effect of CP55940 treatment in GRK5 shRNA-transfected cells compared with vehicle-treated GRK5 shRNA-transfected cells. D, qRT-PCR to show the effect of GRK5 knockdown on GP1a-induced increases in 5-HT2A receptor mRNA levels. **, p 0.01, significant effect of GP1a treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lenti- virustransfectionontheGP1a-inducedup-regulationof5-HT2Areceptors.**, p 0.01, significant effect of GP1a treatment in GRK5 shRNA-transfected cells compared with vehicle-treated GRK5 shRNA-transfected cells. The data rep- resent mean S.E. (error bars) (n 3).

Techniques: Transfection, Control, shRNA, Quantitative RT-PCR, Western Blot, Stable Transfection, Knockdown

FIGURE 6. CB2 and GRK5 are necessary for the CP55940 and GP1a-in- duced increases in 5-HT2A receptor-mediated Ca2 release in CLU213 cells. Cells were stably transfected with either control, CB2, or GRK5 shRNA lentiviral particles as described under “Experimental Procedures.” A, cells sta- bly transfected with control or CB2 shRNA lentiviral particles were pretreated with either vehicle, 1 nM CP55940, 1 nM GP1a, or 15 nM ACEA for 72 h. Cells were then preincubated with 10 nM SB242084 for 20 min and then stimulated with 0.4 nM 5-HT. B, cells stably transfected with control or GRK5 shRNA lenti- viral particles were pretreated with either vehicle, 1 nM CP55940, or 15 nM GP1a. Cells were then preincubated with 10 nM SB242084 for 20 min and then stimulated with 0.4 nM 5-HT. **, p 0.01, significant effect of CP55940 pre- treatment/5-HT stimulation or GP1a pretreatment/5-HT stimulation in con- trol shRNA-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GP1a pretreatment/5-HT stimulation in control shRNA-transfected cells compared with GP1a pretreatment/5-HT stimulation in CB2 or GRK5 shRNA-transfected cells. @@, p 0.01, significant effect of CP55940 pretreatment/5-HT stimulation in control shRNA-transfected cells compared with CP55940 pretreatment/5-HT stimulation in CB2 or GRK5 shRNA-transfected cells. The data represent mean S.E. (error bars) (n 3).

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 6. CB2 and GRK5 are necessary for the CP55940 and GP1a-in- duced increases in 5-HT2A receptor-mediated Ca2 release in CLU213 cells. Cells were stably transfected with either control, CB2, or GRK5 shRNA lentiviral particles as described under “Experimental Procedures.” A, cells sta- bly transfected with control or CB2 shRNA lentiviral particles were pretreated with either vehicle, 1 nM CP55940, 1 nM GP1a, or 15 nM ACEA for 72 h. Cells were then preincubated with 10 nM SB242084 for 20 min and then stimulated with 0.4 nM 5-HT. B, cells stably transfected with control or GRK5 shRNA lenti- viral particles were pretreated with either vehicle, 1 nM CP55940, or 15 nM GP1a. Cells were then preincubated with 10 nM SB242084 for 20 min and then stimulated with 0.4 nM 5-HT. **, p 0.01, significant effect of CP55940 pre- treatment/5-HT stimulation or GP1a pretreatment/5-HT stimulation in con- trol shRNA-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GP1a pretreatment/5-HT stimulation in control shRNA-transfected cells compared with GP1a pretreatment/5-HT stimulation in CB2 or GRK5 shRNA-transfected cells. @@, p 0.01, significant effect of CP55940 pretreatment/5-HT stimulation in control shRNA-transfected cells compared with CP55940 pretreatment/5-HT stimulation in CB2 or GRK5 shRNA-transfected cells. The data represent mean S.E. (error bars) (n 3).

Techniques: Stable Transfection, Transfection, Control, shRNA

FIGURE 7. GRK5 mediates GP1a-induced increases in CB2 phosphoryla- tionandenhanced-arrestin2/ERKinteractioninCLU213cells.Toexam- ine the role of GRK5 in the GP1a-induced increases in CB2 receptor phospho- rylation and -arrestin 2/ERK interaction, cells stably transfected with control of GRK5 shRNA lentiviral particles were treated with either vehicle or 1 nM GP1a for 72 h. A, phosphorylated proteins were separated and detected as described under “Experimental Procedures.” 30 g of isolated phosphoryl- ated protein was used in Western blot detection. **, p 0.01, significant effect of GP1a treatment on CB2 phosphorylation levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lentivirus transfection on the GP1a- induced increases in CB2 phosphorylation. The data represent mean S.E. (n 3). B, CB2 receptor protein levels in whole cell lysate were evaluated by Western blot as described under “Experimental Procedures.” Representative Western blots are shown, and -actin was used as a loading control. The data represent mean S.E. (n 3). C, co-immunoprecipitation was used to exam- inetheroleofGRK5inGP1a-inducedincreasesin-arrestin2/ERKinteraction. Co-immunoprecipitation was conducted as described under “Experimental Procedures.” Negative controls (lanes 9–12) received the same concentration of -arrestin 2 antibody except that the coupling resin was replaced with control agarose resin that is not amine-reactive. All columns were incubated with whole cell lysate (300 g) from vehicle-treated (lanes 1 and 3) or GP1a- treated (lanes 4 and 6) cells. Cell lysate (15 g of protein) was used as an input control (lanes 1–4). The data represent mean S.E. (error bars) (n 4).

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 7. GRK5 mediates GP1a-induced increases in CB2 phosphoryla- tionandenhanced-arrestin2/ERKinteractioninCLU213cells.Toexam- ine the role of GRK5 in the GP1a-induced increases in CB2 receptor phospho- rylation and -arrestin 2/ERK interaction, cells stably transfected with control of GRK5 shRNA lentiviral particles were treated with either vehicle or 1 nM GP1a for 72 h. A, phosphorylated proteins were separated and detected as described under “Experimental Procedures.” 30 g of isolated phosphoryl- ated protein was used in Western blot detection. **, p 0.01, significant effect of GP1a treatment on CB2 phosphorylation levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lentivirus transfection on the GP1a- induced increases in CB2 phosphorylation. The data represent mean S.E. (n 3). B, CB2 receptor protein levels in whole cell lysate were evaluated by Western blot as described under “Experimental Procedures.” Representative Western blots are shown, and -actin was used as a loading control. The data represent mean S.E. (n 3). C, co-immunoprecipitation was used to exam- inetheroleofGRK5inGP1a-inducedincreasesin-arrestin2/ERKinteraction. Co-immunoprecipitation was conducted as described under “Experimental Procedures.” Negative controls (lanes 9–12) received the same concentration of -arrestin 2 antibody except that the coupling resin was replaced with control agarose resin that is not amine-reactive. All columns were incubated with whole cell lysate (300 g) from vehicle-treated (lanes 1 and 3) or GP1a- treated (lanes 4 and 6) cells. Cell lysate (15 g of protein) was used as an input control (lanes 1–4). The data represent mean S.E. (error bars) (n 4).

Techniques: Stable Transfection, Transfection, Control, shRNA, Isolation, Western Blot, Phospho-proteomics, Immunoprecipitation, Concentration Assay, Incubation

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