DSS crosslinking

Crosslinking was performed according to instructions of disuccinimidyl suberate (DSS) crosslinkers (Thermo Scientific) (Palsson-McDermott et al., 2015). Cells were pre-treated 1 h with Shikonin (0.5, 1, 2 μM), and then challenged with LPS (250 ng/ml) and IFN-γ (100 ng/ml) for 24 h, cells were collected and washed two times with ice-cold PBS. Add the DSS solution to a final concentration of 500 nМ, and incubate the reaction mixture for 30 min at room temperature. Add the Quench Solution to a final concentration of 1M Tris-Hcl (pH = 7.5) and incubate the quenching reaction for 15 min at room temperature. Lysates were analyzed by western blot.