GenJet™ protocol was used for B7-H4 overexpression in the HEK293 cell line (Signagen, Frederick, MD, USA). HEK293 cells were plated in a 6-well plate for Western blot analysis or in an 8-well chamber slide for immunofluorescence assay. Lipofectamine® RNAiMAX Reagent protocol (ThermoFisher) was used for B7-H4 interference (RNAi) by short interfering RNAs (siRNAs) in Caki-1 and 786-O cell lines. Cells were plated in 6-well plates for Western blot analysis or in 96-well plates for posterior viability assay; 24 h after plating the cells, the silencing was performed following the RNAiMAX transfection procedure using 20 nM siRNAs. siRNAs for the human B7-H4 gene used were from FlexiTube GeneSolution (GS79679 for VTCN1/B7-H4, Product number: 1027416; SI04365039 (siB7-H4 #1) and SI04346433 (siB7-H4 #2), FlexiTube siRNA Qiagen), and si non-specific (siNS) RNAs and si GAPDH (AM4605; ThermoFisher).
Sigapdh
Manufactured by Thermo Fisher Scientific
The SiGAPDH is a laboratory equipment product designed to perform glyceraldehyde 3-phosphate dehydrogenase (GAPDH) silencing in cell cultures. It serves as a tool for researchers to study gene expression and regulation. The core function of the SiGAPDH is to facilitate the knockdown of the GAPDH gene, which is often used as a housekeeping gene for normalization in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Flexitube sirna, by Qiagen (1 mentions)
Lipofectamine rnaimax reagent protocol, by Thermo Fisher Scientific (1 mentions)
Sictr, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ultraglutamine, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Hiperfect, by Qiagen (1 mentions)
3 protocols using sigapdh
1
Overexpression and RNAi of B7-H4 in Cell Lines
2
Silencing Syndecan-1 in Cardiac Arrhythmia
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Syndecan-1 expression was silenced using siRNA against syndecan-1 (siSyn; cat no. AM16708; Thermo Fisher Scientific, Inc.). The negative control used was a non-targeted siRNA with limited sequence similarity to the targeted gene (siCtr; cat no. AM4611; Thermo Fisher Scientific, Inc.). siRNA against GAPDH was also used (siGAPDH; cat no. AM4631; Thermo Fisher Scientific, Inc.) as a control, The siRNAs were suspended in water to a final concentration of 0.5 µg/µl. To facilitate the transfection, 0.5 µl 50 nM Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was mixed with the siRNA 20 min prior to injection. The control, initial- and late-stage cardiac arrhythmia groups were each divided into four groups as follows: Untreated, siCtr, siSyn and siGAPDH. Each group had six mice. The siRNA was injected into the tail veins of mice at the ages of 4 months (mice developing with initial-stage cardiac arrhythmia) or 7 months (mice developing with late-stage cardiac arrhythmia). The mice were sacrificed 1 month later (at the end of 5 and 8 months, respectively) and cardiac tissue samples were collected for analysis by western blotting.
3
Generation of Stable Cell Lines for Protein Expression
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HeLa and RPE1 FlpIn cells were respectively grown in DMEM and DMEM/F12 supplemented with 8% FBS (Lonza), penicillin/streptomycin (50 μg ml−1), Ultra-glutamine (Sigma; 2 mM), blasticidin (4 μg ml−1) and hygromycin for HeLa (200 μg ml−1) or puromycin for RPE1 (1.6 μg ml−1). 293Ts were grown in DMEM supplemented with 8% FBS (Lonza), penicillin/streptomycin (50 μg ml−1) and Ultra-glutamine (Sigma; 2 mM). Plasmids were transfected using Fugene HD (Roche) for HeLa or Lypofectamin LTX (Invitrogen) for RPE1 according to the manufacturer's instructions. To generate stably integrated HeLa and RPE1 FlpIn cell lines, pCDNA5-constructs were co-transfected with pOG44 recombinase in a 1:9 for HeLa and 1:5 ratio for RPE1 (ref. 55 (link)). Constructs were expressed by addition of 1 μg ml−1 doxycycline for 24 h siHEC1 (custom; Thermo Fisher Scientific; 5′-CCCUGGGUCGUGUCAGGAA-3′) and siGAPDH (Thermo Fisher Scientific; D-001830-01-50) were transfected using HiPerfect (Qiagen) according to manufacturer's instructions.
Cells expressing RFP-MAD2 were obtained through lentiviral transduction and subsequent selection with puromycin (1.6 μg ml−1).
Cells expressing RFP-MAD2 were obtained through lentiviral transduction and subsequent selection with puromycin (1.6 μg ml−1).
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