The following reagents were used: DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt) (Sigma-Aldrich, Saint Louis, MO, USA); morphine hydrochloride (Takeda Pharmaceutical, Tokyo, Japan); pertussis toxin (PTX), an inhibitor of the G protein (Gi, Go and Gt) heterotrimer interaction with receptors (Tocris, Bio-Techne Japan, Tokyo, Japan); Actinomycin D (Act D), a transcriptional inhibitor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); U0126, a potent, selective inhibitor of MEK1 and 2 (Tocris, Bio-Techne Japan, Tokyo, Japan); and Pyridone 6, a potent pan-JAK inhibitor (Tocris, Bio-Techne Japan, Tokyo, Japan). Morphine was dissolved in saline for animal injection. For in vitro experiments, DAMGO, morphine and PTX were diluted with H2O, while the other reagents were diluted with dimethyl sulfoxide.
Actinomycin d act d
Manufactured by Fujifilm
Sourced in Japan
Actinomycin D (Act D) is a laboratory reagent produced by Fujifilm. It is a small molecule that functions as an inhibitor of DNA-dependent RNA synthesis. Act D binds to the DNA template, preventing the initiation of RNA transcription.
Automatically generated - may contain errors
Lab products found in correlation
Damgo, by Merck Group (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Diphenyleneiodonium dpi, by Enzo Life Sciences (1 mentions)
Trichostatin a tsa, by Cayman Chemical (1 mentions)
Rptec complete supplement, by Merck Group (1 mentions)
Dexamethasone (dex), by Fujifilm (1 mentions)
Nmdi14, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
3 protocols using actinomycin d act d
1
Pharmacological Modulation of Opioid Signaling
2
Molecular Mechanisms of Cellular Regulation
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TPA and HRP-conjugated goat anti-rabbit (A6154) and anti-mouse (A4416) IgG were purchased from Sigma-Aldrich Co. (St. Louis, MO). A PKC inhibitor (GF109203X) and actinomycin D (ActD) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cyclopentylidene-(4-(4′-chlorophenyl)thiazol-2-yl)hydrazone (CPTH2) was purchased from Calbiochem (San Diego, CA). 5-(and-6)-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFHDA) and dihydroethidium (DHE) were purchased from Molecular Probes (Eugene, OR). Diphenyleneiodonium (DPI) and garcinol (Gar) were purchased from Enzo Life Sciences Inc. (Farmingdale, NY). Trichostatin A (TSA) was purchased from Cayman Chemical (Ann Arbor, MI). An anti-phospho-PKC (pan) (βII Ser660) rabbit polyclonal antibody (#9371) and normal rabbit IgG (#2729) were purchased from Cell Signaling Technology (Danvers, MA). Anti-actin mouse monoclonal antibody (MAB1501) and anti-acetyl-histone H3 (#06-599) and H4 (#06-598) rabbit polyclonal antibodies were purchased from Millipore Co. (Billerica, MA).
3
Synchronization of Circadian Clock in RPTECs
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Human renal proximal tubular epithelial cells (RPTECs; SA7K clone) were commercially obtained from Sigma-Aldrich. Cells were cultured under 5% CO2 at 37 °C in MEMα supplemented with 5.5% RPTEC Complete Supplement (Sigma-Aldrich), 2.33 mM l -glutamine (Sigma-Aldrich), 28 μM gentamicin (Sigma-Aldrich), and 14 nM amphotericin B (Sigma-Aldrich). For synchronization of the circadian clock in RPTECs, cells were treated with 100 nM DEX (FUJIFILM Wako Pure Chemical Corporation) for 2 h. The medium was replaced with fresh medium, and cells were collected to extract RNA and protein at the indicated time points. For the mRNA stability assay, RPTECs were treated with 5 μM actinomycin D (ActD; FUJIFILM Wako Pure Chemical Corporation), a transcription inhibitor, and cells were collected for RNA extraction at the indicated time points. For splicing analysis, RPTECs were treated with 1 μM NMDI-14 (Merck Millipore). Six or twenty-four hours later, cells were collected to extract RNA or protein, respectively.
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