To block GRP receptors, we used the potent BB2 receptor antagonist RC-3095 (Sigma-Aldrich, 0.9 mM in sterile 0.9% saline) or the non-peptidergic BB2 antagonist PD176252 (Tocris, 0.342 mM in 50% dimethyl sulfoxide (DMSO)). To block VIP receptors, we used the VIP antagonist [D-p-Cl-Phe6, Leu17]-VIP (Tocris, 1 mM in sterile 0.9% saline) or the potent VPAC2 antagonists PG99-465 (Bachem, 1 mM in 0.9% sterile saline). To test the antagonistic properties of RC-3095, we also used porcine GRP (Sigma-Aldrich, 0.3 mM in sterile 0.9% saline). Cocktails of RC-3095 + PG99-465 and of PD176252 + PG99-465 were prepared so that the molarity of each substance was identical when administered alone and in the cocktail. Molarity of antagonists were selected to be about 10x greater than the minimal molarity of GRP and VIP that yielded maximal phase shifts in previous studies (Piggins et al., 1995) . Injections occurred in DD with the aid of night-vision goggles (BG15Alista, Richmond Hill, Ontario, Canada). All manipulations occurred approximately 10 days prior to and following cage changes.
Rc 3095
Manufactured by Merck Group
Sourced in United States
The RC-3095 is a centrifuge that can be used for a variety of laboratory applications. It is designed to separate different components of a liquid sample based on their density and size. The core function of the RC-3095 is to provide a controlled environment for the separation process, allowing users to isolate and extract specific substances from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (2 mentions)
Mdv3100, by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Mycoplasma detection kit, by Southern Biotech (2 mentions)
Lncap, by American Type Culture Collection (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit and anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
3 4 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
β actin antibody, by Bioworld Technology (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Rankl, by ProSpec (1 mentions)
Antibody against β actin, by Cell Signaling Technology (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Hrp conjugated secondary antibody, by Vector Laboratories (1 mentions)
10 protocols using rc 3095
1
Blocking GRP and VIP Receptors to Study Circadian Rhythms
2
Investigating Signaling Pathways in Cancer
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GRP and RC-3095 were purchased from Sigma. AG490 and WP1066 were supplied by Calbiochem and Sigma, respectively. The antibodies for GRP-R and MMP-9, were supplied by Santa Cruz Biotechnology. The antibodies for phospho-JAK2, JAK2, phospho-STAT3, and STAT3 were obtained from Cell Signaling. MMP-2 and β-actin antibodies were purchased from Abcam and Bioworld Technology, respectively. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG were obtained from Thermo Fisher Scientific.
3
Adipogenic Differentiation of 3T3-L1 Cells
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RC-3095 and GRP were provided by Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and calf serum (CS) were obtained from Gibco Life Technologies (Grand Island, NY, USA). 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and Oil Red O solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-[4-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich. Rabbit polyclonal GRP-R and rabbit monoclonal CREB antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively. β-actin antibody was purchased from Bioworld Technology (St. Louis Park, MN, USA).
4
Intrathecal Delivery of Neuropeptides
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Naltrexone HCl, opioid-related neuropeptides (National Institute on Drug Abuse, Bethesda, MD, USA), GRP, nor-binaltorphimine HCl (Tocris Bioscience, Minneapolis, MN, USA) and RC-3095 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in sterile water. The neuropeptide alone or combined with the antagonist was delivered intrathecally at a total volume of 1 mL. A detailed description of the lumbar intrathecal drug delivery has been previously described8 (link)34 (link). The neuropeptide was delivered intrathecally with a 10-day inter-injection interval as previous studies did8 (link)25 (link).
5
Establishing Androgen-Resistant Prostate Cancer Models
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The human prostate carcinoma cell line LNCaP and 22RV1 were obtained from the ATCC (Manassas, VA). LNCaP-MDV cells were generated by treating LNCaP cells with MDV3100 (10−5M) for more than 3 months (named LNCaP-MDV) and maintained in the regular culture medium with MDV3100 (10−5M) for further studies. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in the air and were tested for contamination within the past 6 months using a Mycoplasma Detection Kit (Southern Biotech). Cell lines were routinely cultured in RPMI 1640 (Gibco-BRL) medium containing 5% fetal calf serum (FBS) (Hyclone), 0.1% Insulin-Transferrin-Selenium (ITS) and 0.1% Glutamine (Gibco-BRL). The following reagents were purchased for in vitro and in vivo experiments: RC3095 (a selective GRP-R antagonist; Sigma-Aldrich) and MDV3100 (an AR antagonist; Sigma).
6
Macrophage Activation and Signaling Pathways
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Macrophage colony-stimulating factor (M-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA) and receptor activator of nuclear factor kappa-Β ligand (RANKL) from ProsPec (Rehovot, Israel). GRP and RC-3095 were purchased from Sigma (St. Louis, MO, USA). The antibodies against GRP and GRPR were from Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The antibody against β-actin was from Cell Signaling (Danvers, MA, USA). Mayer’s hematoxylin was from Dako (Glostrup, Denmark). HRP-conjugated secondary antibody and DAB were from Vector Labs (Burlingame, CA, USA). Lipopolysaccharide (LPS) from P. gingivalis was purchased from Sigma Aldrich (St. Louis, MO, USA).
7
Intrathecal Delivery of Gastrin-Releasing Peptides
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Gastrin-releasing peptide peptide (R&D Systems, MN) and RC-3095 (Sigma-Aldrich, MO)
were dissolved in sterile water. Under adequate inhalation anesthesia with sevoflurane
(3%–5%, in air), the vertebral arch of the L5 vertebra was resected and a 5 mL soft tube
(SILASTIC laboratory tubing, O.D.: 0.64 mm; Dow Corning Corporation, MI, USA) filled with
saline solution was inserted. The tube was inserted 10 mm into the subarachnoid space
(toward the head). For a single intrathecal injection of GRP, the end of the soft tube was
ligated and fixed subcutaneously. The next day, under sevoflurane inhalation anesthesia,
the ligated tube was cut and GRP was administered using a Hamilton syringe. Furthermore,
0.03 nmol and 0.3 nmol doses of GRP were administered (diluted in saline at 8.5 μg/mL and
85 μg/mL, respectively).19 (link),20 (link) For
intrathecal administration of RC-3095 for 1 day, a pump (MINI-OSMOTIC PUMP MODEL 2001D;
DURECT Corporation, CA) was connected to a soft tube and the pump was held subcutaneously
with sutures. The dosages of RC-3095 were 0.3 nmol and 3 nmol per day (diluted in saline
at 14.2 ng/mL and 142 ng/mL, respectively), and the solutions were injected using a
mini-pump.
were dissolved in sterile water. Under adequate inhalation anesthesia with sevoflurane
(3%–5%, in air), the vertebral arch of the L5 vertebra was resected and a 5 mL soft tube
(SILASTIC laboratory tubing, O.D.: 0.64 mm; Dow Corning Corporation, MI, USA) filled with
saline solution was inserted. The tube was inserted 10 mm into the subarachnoid space
(toward the head). For a single intrathecal injection of GRP, the end of the soft tube was
ligated and fixed subcutaneously. The next day, under sevoflurane inhalation anesthesia,
the ligated tube was cut and GRP was administered using a Hamilton syringe. Furthermore,
0.03 nmol and 0.3 nmol doses of GRP were administered (diluted in saline at 8.5 μg/mL and
85 μg/mL, respectively).19 (link),20 (link) For
intrathecal administration of RC-3095 for 1 day, a pump (MINI-OSMOTIC PUMP MODEL 2001D;
DURECT Corporation, CA) was connected to a soft tube and the pump was held subcutaneously
with sutures. The dosages of RC-3095 were 0.3 nmol and 3 nmol per day (diluted in saline
at 14.2 ng/mL and 142 ng/mL, respectively), and the solutions were injected using a
mini-pump.
8
Investigating Bombesin-Mediated Signaling
9
Bone Cell Signaling Pathway Analysis
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RC-3095 and GRP were purchased from Sigma (St. Louis, MO, USA). The antibodies against GRP and GRP receptor were supplied by Abcam (Cambridge, UK) and Santa Cruz Biotechnology, (Santa Cruz, CA, USA), respectively. The antibodies against phospho-Smad1/5, Runx2, phospho- ERK, ERK, phospho-p38, p38, total/cleaved caspase-3, total/cleaved caspase-9, Bcl2 and Bad were obtained from Cell Signaling (Danvers, MA, USA). Calponin and β-actin antibodies were purchased from Abcam and Bioworld Technology (St. Louis Park, MN, USA), respectively. Smad1/5, horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
10
Prostate Cancer Cell Line Maintenance
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The human prostate carcinoma cell line LNCaP and 22RV1 were obtained from the ATCC (Manassas, VA). Cells were maintained at 37°C in a humidi ed atmosphere of 5% CO 2 in the air and were tested for contamination within the past 6 months using a Mycoplasma Detection Kit (Southern Biotech). Cell lines were routinely cultured in RPMI 1640 (Gibco-BRL) medium containing 5% fetal calf serum (FBS) (Hyclone), 0.1% ITS and 0.1% Glutamine (Gibco-BRL). The following reagents were purchased for in vitro and in vivo experiments: RC3095 (Sigma-Aldrich) and MDV3100 (Sigma).
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