SMAD3 (s8401 and s8402) silencer select siRNAs were purchased from Life Technologies. siRNA transfection was performed using Lipofectamine RNAiMAX (Life Technologies). For each well treated with the SMAD3 siRNA or scrambled control (Life technologies, #4390843), the final concentration was 20 nM. HCASMCs were seeded in 6 well plates and grown to 75% confluence before siRNA transfection. HCASMCs were transfected with the SMAD3 siRNA or scrambled control for 12 hours and subsequently collected and processed for RNA isolation after 48 hrs of transfection using the RNeasy kit (Qiagen). For the SMAD3 overexpression study, HCASMCs were transduced with 5ug of pRK5F-SMAD3 cDNA (Addgene plasmid# 12625) or control pCDNA3.1 DNA (ThermoFisher Scientific, plasmid# V79020) using the Amaxa Basic Nucleofector kit for primary mammalian smooth muscle cells (Lonza #VPI-1004) at a density of 1x106 cells per 100 μL sample using Nucleofector Program U-025. Cells were changed to medium with supplements 24hrs after transfection and cultured for an additional 48 hrs. The transduction efficiency was assessed by transducing HCASMCs with 2 μg of pmax-GFP cDNA and quantifying the percentage of GFP positive cells by quantitative fluorescence microscopy.
Smad3 sirna
Manufactured by Thermo Fisher Scientific
Sourced in Japan
Smad3 siRNA is a synthetic small interfering RNA (siRNA) molecule designed to target and silence the expression of the Smad3 gene. Smad3 is a key intracellular signaling protein involved in the transforming growth factor-beta (TGF-β) signaling pathway. The Smad3 siRNA can be used in cell-based assays and research applications to study the role of Smad3 in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Amaxa basic nucleofector kit for primary mammalian smooth muscle cells, by Lonza (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Bix 01294, by Merck Group (1 mentions)
Tgfb1, by R&D Systems (1 mentions)
Tgf b1, by Merck Group (1 mentions)
Nonsense sirna, by Thermo Fisher Scientific (1 mentions)
Vegf elisa kit, by Elabscience (1 mentions)
Nur77 sirna, by Thermo Fisher Scientific (1 mentions)
Scrambled sirna, by Thermo Fisher Scientific (1 mentions)
Nrk 49f, by American Type Culture Collection (1 mentions)
Nrk 52e, by American Type Culture Collection (1 mentions)
Tgf β1, by Abcam (1 mentions)
Nrk 52e cells, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Anti tgf β1 antibody, by Abcam (1 mentions)
7 protocols using smad3 sirna
1
Silencing and Overexpressing SMAD3 in HCASMCs
2
Evaluating BIX01294 Effects on TGF-β1 Response
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Cell culture was performed as described previously. 52 Smad3 siRNA was purchased from Life Technologies. Cells were transfected using Lipofectamine 2000 Reagent (Life Technologies) according to the manufacturer's protocol. After incubation with transfection complexes overnight, the medium was changed, and the cells were stimulated with 5 ng/ml TGF-b1 (R&D Systems) for 24 h. To determine the effects of BIX01294 on cultured cell lines, 2 mmol/l BIX01294 (Sigma-Aldrich) was incubated with subconfluent cells 1 h before TGF-b1 stimulation. Cells were then exposed to TGF-b1 (5 ng/ml) with or without BIX01294 for 24 or 48 h.
3
HVOX Fibroblast Cell Line Knockdown
4
Silencing HIF-1α in THP-1 Cells
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siRNA specific to HIF-1α was selected as described previously and purchased from Sigma (Suffolk, UK) together with a mutated form of siRNA (called random siRNA, which was used as negative control) [24 (link)]. We employed a HIF-1α-specific siRNA target sequence (ugu gag uuc gca ucu uga u dtdt) localised at position 146 bases downstream of the HIF-1α start codon. Smad3 siRNA was a commercially available reagent purchased from Ambion (ID 107876) through Thermo Fisher Scientific. Random (mutated) siRNA used as a negative control in all the knockdown experiments had the following sequence: uac acc guu agc aga cac c dtdt. siRNAs were transfected into THP-1 cells using DOTAP transfection reagent according to the manufacturer's protocol. Successful HIF-1α knockdown was verified by assessing HIF-1 DNA-binding activity.
5
Investigating EMT Markers in Cell Signaling
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Rabbit anti-human restructuring anti-Smad3 (Abcam USA, ab40854), rabbit anti-human NF-κB p65 antibody C-20 (Santa Cruz Biotechnology USA, sc-372), rabbit anti-human TGF-β1 antibody V (Santa Cruz Biotechnology USA, sc-146), mouse monoclonal antibody TNF-α (Abcam USA, ab1793), and rabbit anti-human ZO-1 antibody (Abcam USA, ab221547) were used. CCK8 (Cell Counting Kit-8) kit (Tongren Chemical, Japan), VEGF-ELISA kit (Elabscience), Smad3-siRNA (Invitrogen), and EMT detection markers E-cadherin and vimentin were purchased from Beijing Keystone Life Technology. All other reagents were used at an analytical grade.
6
Silencing Key Signaling Regulators
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Smad2, Smad3, Nur77 and scrambled small interfering RNAs (siRNAs) were ordered from Bioneer Technology (Daejon, Republic of Korea). The sense sequence for the siRNAs was as follows: Smad2 siRNA: 5′-GCAGAUUUUCCUUGUAGAAdTdT-3′; Smad3 siRNA: 5′-GCGUAUAGGUGAUGUACAGdTdT-3′; Nur77 siRNA: 5′-UCCCUGGCUUCAUUGAGCUUdTdT-3′; scrambled siRNA: 5′-CCUACGCCACCAAUUUGGUdTdT-3′. MA-10 and HEK293T cells were transfected with 20 nM siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
7
Regulation of TGF-β1 Signaling in NRK Cells
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NRK-52E and NRK-49F cells were purchased from the American Type Culture Collection (Manassas, VA). These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were washed and growth-arrested for 24 hours in DMEM including 0.1% FBS before each stimulation. NRK-52E and NRK-49F cells were stimulated with TGF-β1 at the indicated doses and times. NRK-52E cells were treated with Smad3 siRNA (Invitrogen, Carlsbad, CA) using Lipofectamine 2000 Reagent (Invitrogen) in accordance with the product protocol. After incubation with transfection complexes for 6 hours, the medium was changed, and the cells were stimulated with 1.0 ng/mL TGF-β1 (R&D Systems) for 24 hours. For Hira siRNA (Invitrogen), incubation with transfection complexes was for 24 hours. To counteract the effects of TGF-β1 on cultured cells, subconfluent cells were incubated with 2.0 μg/mL anti-TGF-β1 antibody (Abcam, Cambridge, UK) at the same time as TGF-β1 stimulation. Cells were then exposed to TGF-β1 (1.0 ng/mL) with anti-TGF-β1 antibody for 24 hours.
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