A change in CRAMP levels was measured in neonatal mouse cardiomyocytes (NMCMs) while functional and mechanism experiments were performed in neonatal rat cardiomyocytes (NRCMs). NMCMs and NRCMs were isolated and cultured, as previously reported 9 (link). Cardiomyocyte hypertrophy was induced by Ang Ⅱ or PE treatment. After starvation with serum-free DMEM (Gibco, USA) for 6-8 h, cardiomyocytes were treated with Ang Ⅱ (1 μM) or PE (100 μM) for 48 h. Cardiomyocytes were simultaneously treated with CRAMP (0.1 mg/L, Innovagen AB) for 48 h. Betulinic acid (BA, Tocris), an agonist of NF-κB was used and NRCMs were treated with rCRAMP (rat homolog for human LL-37) in the presence or absence of Betulinic acid (BA, 20 μM, Tocris), for 48 h.
Betulinic acid
Manufactured by Bio-Techne
Sourced in United States
Betulinic acid is a natural pentacyclic triterpene compound isolated from the bark of various plant species. It is a chemical reference standard used for analytical purposes in research and development.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Neun antibody, by Merck Group (1 mentions)
Real time pcr rt pcr primers, by Qiagen (1 mentions)
Antibodies against, by Fujifilm (1 mentions)
Antibodies against tgr5, by Abcam (1 mentions)
Glast pe, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lncap, by Thermo Fisher Scientific (1 mentions)
22rv1 crl 2505, by American Type Culture Collection (1 mentions)
Lactacystin, by Bio-Techne (1 mentions)
Cd 1 mice, by Charles River Laboratories (1 mentions)
Mg132, by Bio-Techne (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Normacin, by InvivoGen (1 mentions)
Sb525334, by Bio-Techne (1 mentions)
Mithramycin a, by Bio-Techne (1 mentions)
5 protocols using betulinic acid
1
CRAMP Modulates Cardiomyocyte Hypertrophy
2
Immunohistochemical Analysis of Microglia
3
Prostate Cancer Cell Culture Protocol
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Human prostate cancer cell lines LNCaP.FGC (CRL-1740) and 22Rv1 (CRL-2505) were purchased from American Type Culture Collection (Manassas, VA, USA). LNCaP and 22Rv1 cells were grown and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red (Life Technologies, Inc., Grand Island, NY, USA) supplemented with l -glutamine and 10% fetal bovine serum (FBS) (Life Technologies). To minimize the influence of hormones present in normal FBS on our experiments, charcoal stripped FBS (Cat. No. F6765, Sigma-Aldrich, St. Louis, MO, USA) was used in all experiments involving 22Rv1. However, normal FBS was used in LNCaP cells whose growth was significantly compromised in charcoal stripped FBS. The expression plasmids which contain full-length AR (pEGFP-AR-FL) and AR-V7 (pEGFP-AR-V7) were kindly provided by Dr. J. Luo (Johns Hopkins University, Baltimore, MD, USA). The cDNAs encoding AR-V7 and full-length AR were inserted into the pEGFP-C3 vector to express the EGFP-AR-V7 and EGFP-AR-FL fusion protein respectively [8 (link),37 (link)]. Melatonin (Cat. No. M5250) and luzindole (Cat. No. L2407) were purchased from Sigma-Aldrich. Betulinic acid, which is a NF-κB activator, was obtained from Tocris (Bristol, UK).
4
Isolation and Treatment of Cortical Neurons
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Timed pregnant CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Cerebral cortical neurons were prepared as described previously [21 (link)]. Briefly, cerebral cortices from embryos at E16 were aseptically dissected and plated at 3.5 × 105 cells per well on polyethyleneimine-coated 6-well plates. Neurons were cultured in Neurobasal medium supplemented with 2 mM L-glutamine, B27 and antibiotics (Invitrogen). The media was replaced with Neurobasal supplemented with B27 minus antioxidants, glutamine, and antibiotics on the third day in vitro (DIV 3). Treatment of neurons was conducted at DIV 14. The following pharmacological treatments were used: MG132 (20 μM); lactacystin (50 μM); or betulinic acid (20 μg/mL) (all the reagents were from Tocris Biosciences, Minneapolis, MN, USA). At the end of the treatments, cells were washed in Phosphate Buffered Saline (PBS) and lysed in ice-cold lysis buffer supplemented with protease inhibitor cocktail (Sigma) for immunoblotting.
5
Immortalized Leiomyoma and Myometrial Cells
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Patient-matched leiomyoma and myometrial primary cells were isolated and immortalized as described elsewhere by our laboratory (44) .
For UPA studies, patient-matched leiomyoma and myometrial cells were plated in six-well dishes in Dulbecco's minimum essential medium/F12 media (Thermo Fisher Scientific) with 10% fetal bovine serum (Hyclone) and 1Â Normacin (Invivo Gen). Cultures were grown to 30%-40% confluence before treatment with 10 À9 M through 10 À7 M UPA (Sigma-Aldrich). Control cells were treated with dimethyl sulfoxide alone.
For inhibitor studies, immortalized leiomyoma cells were grown to 70%-80% confluence before exposure to serum-free media (Dulbecco's minimum essential medium/F12 media containing 1Â Normacin and 0.1% bovine serum albumin) for 48 hour. Treatment with inhibitors was carried out in serum-free media as follows: TGF-bRI kinase inhibitor, SB525334 (Tocris, 1 mM) for 2 hours; SP-1 specific inhibitors, mithramycin A (Tocris, 10 nM) for 2-4 hours, tolfenamic acid (Tocris, 25 mM) for 2 hours, and betulinic acid (Tocris, 10 mM) for 2 hours; MAP kinase pathway (MEK1/MEK2) inhibitor U0126 (Calbiochem, 1 mM) for 2 hours. Treatment with inhibitors was followed by exposure to either TGF-b1 (Millipore, 5 ng/mL) or TGF-b3 (Millipore, 5 ng/mL) for 24 hours at which time cells were collected for RNA isolation.
For UPA studies, patient-matched leiomyoma and myometrial cells were plated in six-well dishes in Dulbecco's minimum essential medium/F12 media (Thermo Fisher Scientific) with 10% fetal bovine serum (Hyclone) and 1Â Normacin (Invivo Gen). Cultures were grown to 30%-40% confluence before treatment with 10 À9 M through 10 À7 M UPA (Sigma-Aldrich). Control cells were treated with dimethyl sulfoxide alone.
For inhibitor studies, immortalized leiomyoma cells were grown to 70%-80% confluence before exposure to serum-free media (Dulbecco's minimum essential medium/F12 media containing 1Â Normacin and 0.1% bovine serum albumin) for 48 hour. Treatment with inhibitors was carried out in serum-free media as follows: TGF-bRI kinase inhibitor, SB525334 (Tocris, 1 mM) for 2 hours; SP-1 specific inhibitors, mithramycin A (Tocris, 10 nM) for 2-4 hours, tolfenamic acid (Tocris, 25 mM) for 2 hours, and betulinic acid (Tocris, 10 mM) for 2 hours; MAP kinase pathway (MEK1/MEK2) inhibitor U0126 (Calbiochem, 1 mM) for 2 hours. Treatment with inhibitors was followed by exposure to either TGF-b1 (Millipore, 5 ng/mL) or TGF-b3 (Millipore, 5 ng/mL) for 24 hours at which time cells were collected for RNA isolation.
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