OK371-gal4 (motor neuron specific driver) was used to overexpress Nups (Nup62 OE, Nup214 OE, Nup43 OE) or eGFP (control), and climbing assays were performed as previously described (Anderson et al., 2018 (link)). Additionally, Elav-GeneSwitch (ElavGS) was used to conditionally express Nup62 RNAi or Nup214 RNAi in neuronal cells on normal cornmeal food. Adult progeny were subjected to repeated trauma as described (Anderson et al., 2018 (link)), and flies that survived after 24 hr were placed on food treated with ethanol (-RU486) or RU486 (+ RU486, Cayman Chemical) for 20 days before motor assay. Adult flies were anesthetized with CO2 after 20 days, transferred to vials, and allowed to recover for 30 min. Flies were knocked to the bottom of vials by tapping against the bench three times, and a video camera was used to record flies climbing up the walls. The percentage of flies that climbed 4 cm in 20 s as well as the velocity (cm/s) of each individual fly was quantified and analyzed using GraphPad Prism six software. Three experimental replicates were performed for each group.
Ru486
Manufactured by Cayman Chemical
Sourced in United States
RU486 is a synthetic steroid compound used in laboratory research. It functions as a progesterone receptor antagonist. This product is intended for use in scientific investigations and should be handled with appropriate safety precautions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Progesterone, by Merck Group (3 mentions)
Aldosterone, by Merck Group (2 mentions)
Triamcinolone acetonide, by Fujifilm (2 mentions)
Dexamethasone sodium phosphate, by Fujifilm (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Cortisol, by Merck Group (2 mentions)
11 deoxycortisol, by Merck Group (2 mentions)
17a hydroxyprogesterone, by Merck Group (2 mentions)
17 20b 21 tri hydroxy progesterone, by Merck Group (2 mentions)
20b hydroxy progesterone, by Merck Group (2 mentions)
17 20b dihydroxyprogesterone, by Merck Group (2 mentions)
11 deoxycorticosterone, by Merck Group (2 mentions)
Corticosterone, by Merck Group (2 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
Ly294002, by Fujifilm (1 mentions)
U0126, by Promega (1 mentions)
Paraquat, by Merck Group (1 mentions)
Anti bax, by BD (1 mentions)
Anti bip grp78, by BD (1 mentions)
29 protocols using ru486
1
Nup Overexpression and Knockdown in Motor Neuron Climbing Assay
2
Müller Glial Cell Line Characterization
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The human Müller glial cell line Moorfields/Institute of Ophthalmology‐Müller 1 (MIO‐M1) was provided from Dr G. Astrid Limb (UCL Institute of Ophthalmology).10 The cells were cultured in DMEM containing 10% FBS (Thermo Fisher Scientific). Twenty‐four hours after transfection, the composite transfection mixture was removed and replaced with 1% FBS/DMEM for 24 hours, followed with recombinant protein and reagent treatments before each assay. Recombinant human IL‐1β proteins were purchased from R&D systems. Aldosterone and streptozotocin (STZ) were from Sigma‐Aldrich. RU486 was from Cayman Chemical. Dexamethasone sodium phosphate, triamcinolone acetonide and LY294002 (PI3K inhibitor) were from FUJIFILM Wako Pure Chemical Corporation. U0126 (ERK1/2 inhibitor) was from Promega. Reagents were dissolved in either PBS, ethanol (final concentration < 0.1%) or dimethyl sulfoxide (DMSO, final concentration < 0.1%).
Specific siRNAs against dual specificity phosphatase (DUSP)1 (hs.Ri.DUSP1.13.3), TSC22 domain family member (TSC22D)3 siRNA (hs.Ri.TSC22D3.13.1) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L. Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.
Specific siRNAs against dual specificity phosphatase (DUSP)1 (hs.Ri.DUSP1.13.3), TSC22 domain family member (TSC22D)3 siRNA (hs.Ri.TSC22D3.13.1) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L. Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.
3
RU486 Feeding Regimen in Drosophila
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RU486 (Cayman Chemical Company Cat. No. 10006317) was added to standard fly food at a final concentration of 200 µm. For larval experiments, eggs were laid directly onto RU-containing food. For adult gut experiments, eggs were laid on and developed on standard food, and adults were transferred to RU-containing food for the indicated time.
4
Drosophila Rearing and Transgene Control
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All flies were cultured on standard cornmeal medium at 25°C under 12 h light, 12 h dark cycles, except for flies analyzed in the temperature sensitive experiment presented in Fig 3 . These flies were cultured in one of three regimens: at 18°C, at 29°C, and at 18°C until eclosion and then at 29°C thereafter. For steroid mediated UAS-transgene control using the Gene-Switch driver, flies were fed food containing 200μM RU-486 (Mifepristone, Cayman Chemicals, Ann Arbor MI). Staging of pupae and MARCM clone induction was performed as previously described [37 (link), 38 (link)]. Unless otherwise noted, adult male flies of indicated ages were used for experiments.
5
Fly Treatment with RU486, Oltipraz, and Paraquat
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For RU486 treatment, 250 μl 80% ethanol ± 5 mM RU486 (Cayman Chemical Company) was added to the surface of the fly food and left to dry overnight in a chemical hood. 20 flies were then added to each vial and fed on RU486 food for 6 days by switching to new RU486 food every 2 days. For oltipraz treatment, flies were kept on food supplemented with 1 mM oltipraz for 72 hours before microscopy or RNA extraction. For paraquat treatment, flies were starved for 2 hours and then kept for 12 hours in vials containing a piece of filter paper, which was soaked with 5% sucrose ±25 mM paraquat (Sigma-Aldrich).
6
RU486 Administration in Rodent Model
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RU486 (cayman) was dissolved in sesame oil at 10 mg/mL and administered (i.p, 50 mg/kg/24 h) at 10 dpi. The respective diluents were given to the control animals at the same time.
7
Investigating Apoptosis Signaling Pathways
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Anti-Bak, anti-BiP/Grp78, anti-Bax, and anti-ERK monoclonal antibodies were from BD Bioscience; anti-PERK, anti-Bcl-xL, anti-Bim, Anti-Bak, anti-Bax, anti-eIF2α, anti-phospho-eIF2α, anti-phospho-ERK, anti-cleaved caspase 6 (cCas6) and anti-cleaved poly(ADP-ribose) polymerase (cPARP) antibodies were from Cell Signaling Technology; and anti-GAPDH antibodies were from GeneTex. Anti-SubAB antibodies were prepared as previously described6 (link). PKC inhibitor Gö6976 and ERK inhibitor U0126 were obtained from LC Laboratories. SB23590 was from WAKO. PKC activators (PMA and prostratin), Thapsigargin, and PKC inhibitor Gö6983 were from Sigma Aldrich. Bryostatin 1 was from Santa Cruz Biotechnology. PKC inhibitor Bisindolylmaleimide II (Bis II) was from ALEXIS Biochemicals. PKC activator Ingenol-3-angelate (I3AG), SP600125, and steroid antagonist RU486 were from Cayman Chemical. Dexamethasone sodium phosphate (Dx) was from FujiPhama. Prednisolone (P) was from SHIONOGI & CO., Ltd. Methylprednisolone Succinate Na (MP) was from Sawai Pharmaceutical Co., Ltd. Hydrocortisone (HC) was from Taisho Pharmaceutical Co., Ltd.
8
Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) Cell Line
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The human Müller glial cell line Moorfields/Institute of Ophthalmology‐Müller 1 (MIO‐M1) was provided from Dr G. Astrid Limb (UCL Institute of Ophthalmology, London, United Kingdom).14 The cells were cultured in DMEM containing 10% fetal bovine serum (Thermo Fisher Scientific). For hypoxic exposure, cells were cultured in a gas mixture composed of 1% O2, 5% CO2 and 94% N2. Aldosterone and streptozotocin were from Sigma‐Aldrich. RU486 and MG132 were from Cayman Chemical. Dexamethasone sodium phosphate, triamcinolone acetonide and actinomycin D were from FUJIFILM Wako Pure Chemical Corporation.
Specific siRNAs against TSC22D3 (hs.Ri.TSC22D3.13.1), DUSP1 (hs.Ri.DUSP1.13.3) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L.11 Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.
Specific siRNAs against TSC22D3 (hs.Ri.TSC22D3.13.1), DUSP1 (hs.Ri.DUSP1.13.3) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L.
9
Inducible Crif1 Knockout in Mice
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Crif1fl/fl mice were generated as previously described [10 (link)], and were crossed with KRT14-CreERT2 (The Jackson Laboratory, Bar Harbor, Maine) or KRT15-CrePR mice (generously provided by Dr. George Cotsarelis, Philadelphia, PA). For induction of knockout, mice were shaved at P21 and topically treated with 4-hydroxy tamoxifen (4-OHT) or RU486 (Cayman, Item No. 10006317, 200 μl of 5 mg/ml in acetone) during first telogen (P22-P26). And hair growth was examined around P35-P44.
For depilation-induced anagen, RU486 (200 μl of 5 mg/ml in corn oil) was intraperitoneally injected daily for 10 times during 2 weeks (P35-P48). The left back skin were depilated at P49 and right back skin was depilated at P56 using hair removal wax. Hair growth was examined at P63.
All experiments were performed in accordance with institutional guidelines and approved by Chungnam National University institutional animal care and use committee (IRB CNU-00654). Mice were maintained in conventional condition with food and water ad libitum and monitored daily to minimize animal suffering. Mice were sacrificed using CO2 gas.
For depilation-induced anagen, RU486 (200 μl of 5 mg/ml in corn oil) was intraperitoneally injected daily for 10 times during 2 weeks (P35-P48). The left back skin were depilated at P49 and right back skin was depilated at P56 using hair removal wax. Hair growth was examined at P63.
All experiments were performed in accordance with institutional guidelines and approved by Chungnam National University institutional animal care and use committee (IRB CNU-00654). Mice were maintained in conventional condition with food and water ad libitum and monitored daily to minimize animal suffering. Mice were sacrificed using CO2 gas.
10
Feeding RU486 to Drosophila
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RU486 (Cayman Chemical Company Cat. No. 10006317) was added to standard fly food at a final concentration of 200 µm. For larval experiments, eggs were laid directly onto RU-containing food. For adult gut experiments, eggs were laid on and developed on standard food, and adults were transferred to RU-containing food for the indicated time.
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