TransIT-TKO Transfection Reagent

Protocol tips

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	siRNA concentration-20nM Mix siRNA:reagent mixture and incubate at room temperature for 15–30 minutes. Add mixture to cells and incubate for 48hours.

Protocol tips
siRNA concentration-20nM Mix siRNA:reagent mixture and incubate at room temperature for 15–30 minutes. Add mixture to cells and incubate for 48hours.

Publication protocol

Caspase-2 knockdown by siRNA has been previously described.45 Two different sets of siRNA (purchased from GenePharma, Shanghai, China) were used to minimize any potential off-target effects, with the following sequences; CASP2 siRNA-1 (5′-ACAGCUGUUGUUGAGCGAAdTdT-3′), CASP2 siRNA-2 (5′-GUUGUUGAGCGAAUUGUUATT-3′), control siRNA (5′-UAAGGCUAUGAAGAGAUACTT-3′). All transfections were carried out using Trans IT-TKO transfection reagent (Mirus, Madison, WI, USA) for 48 h according to manufacturer's instructions. Briefly, U2OS cells (1 × 105) were seeded into 60 mm dishes in 2 ml complete medium and allowed to adhere for 16–24 h. 2.5 μl (20 μm siRNA) and 3 μl of TransIT-TKO siRNA transfection reagent (Mirus) were diluted to 100 μl in Opti-MEM (Sigma-Aldrich) and incubated for 15 min at room temperature. The diluted siRNA and transfection reagent were combined and incubated for a further 15 min at room temperature and then overlaid onto cells. After 30 h incubation at 37 °C in the transfection complexes, the media was replaced and cells were allowed to recover for 12 h before treatment with BI 2536 (100 nm) for 48 h and harvesting for protein analysis. Knockdown experiments were repeated at least three times and the results statistically analysed

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