Silencing of IKKγ by siRNA

In order to achieve 30% confluence at the time of transfection, 1 day before, the cells were seeded without antibiotics. IKKγ siRNA was synthesized in vitro by Silencer™ siRNA Construction Kit (Thermo Fischer, Milan, IT) with the following oligomers:

Sense 5′-AAAAGATTGTGATGGAGACCGCCTGTCTC-3′;

Antisense 5′-AACGGTCTCCATCACAATCTTCCTGTCTC-3′.

siRNA and Lipofectamine™ 2000 (Invitrogen, Milan, Italy) were diluted in Opti-MEM I (Gibco, Milan, Italy) without serum according manufacturer instruction. Cells were transfected with 100 nM of IKKγ siRNA and were incubated at 37°C in a CO₂ incubator. Western blot analysis at 24-48-72 h showed almost no IKKγ protein in silenced cells; only a faint band can be barely detected, indicating presence of few un-transfected cells (Supplementary Figure 1A). All the experiments were performed at 48 h and silencing efficiency was evaluated in w/b as showed in Supplementary Figures 1A,B.