siRNA p65 / WNK1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 5×10^5 cells/well	Final concentration - 80 nM. Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Replace medium after 6h and re-incubate cells for 1–2 days at 37°C.

Upstream tips
Seed 5×10^5 cells/well
Protocol tips
Final concentration - 80 nM. Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Replace medium after 6h and re-incubate cells for 1–2 days at 37°C.

Publication protocol

The NF-κBp65 siRNA, PUMA siRNA and Slug siRNA specific and negative control (NC) were purchased from Dharmacon (Lafayette, CO, USA). Just before transfection, the cells were cultivated in RPMI-1640 medium free of serum and antibiotics. siRNA transfection (at a final concentration of 80 nM in all experiments) was performed using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Briefly, siRNAs and lipofectamine (4 µl/ml of transfection medium) were diluted in Opti-MEM I Reduced Serum Medium (Invitrogen) separately and incubated for 10 min at room temperature. The diluted solutions were then mixed and incubated for 20 min at room temperature. Subsequently, the mixtures were added to each well containing cells and medium. Moreover, the treated cells with only the transfection reagent were considered as a blank control. The cell culture plates were then incubated for 6 h at 37˚C in a CO2 incubator. Following on, RPMI-1640 medium containing FBS (final FBS concentration of 15%) was added, with cells being incubated under the above mentioned conditions. To evaluate the effects of siRNAs on gene silencing, transfections (5×105 cells/well) were performed in 6-well cell culture plates for 24-48h. The suppression of NF-κBp65, PUMA and Slug expression was then assessed by Western blotting.G418 (400 ng/ml) (Life Technologies, Carlsbad, CA, USA) was added to the medium after 48h of transfection, and the cells were cultured for two weeks to permit selection

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