On targetplus non targeting sirna 2

HEK-293T cells were seeded into 6-well plates (3 × 10⁵ cells per well) for transfection on the following day. Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with previously published plasmids [96 (link)], and then harvested 24 h post-transfection for immunoblotting. For siRNA transfections, 494H cells were seeded into 6-well plate (1.6 × 10⁴ cells per well) in antibiotic free media. The following day cells in each well were transfected with 50 nM of On-Targetplus non-targeting siRNA #2 (Thermo Fisher Scientific) or SMART pool: On-Targetplus mouse RIPK1 siRNA (Thermo Fisher Scientific) using 2 μl of DharmaFECT-2 transfection reagent (Thermo Fisher Scientific). Cells were incubated with transfection mixture for 8 h, before replacing media. Transfected cells were cultured for 48 h, and then harvested for immunoblotting and cell death assays.

In a 6-well plate, 30 pmol of indicated siRNAs per 2.5 × 10⁵ C4-I cells/well (ON-TARGETplus Non-targeting siRNA #2, ON-TARGET smartpool for RIPK1 or RIPK3, all from Thermo Scientific, Bonn, Germany) were transfected with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's guidelines. Forty-eight hours later, protein amounts were analyzed by Western blot analysis or used in assays.