In vitro ubiquitination kit assays

Ubiquitination assays were performed according to the instruction of the manufacturer (Boston Biochem, USA). Briefly, the following components were successively added into 0.5 ml polypropylene tubes on ice: 9 μl dH₂O, 3 μl 10× reaction buffer, 3 μl 10× rS5a, cell lysates or rMyD88 substrate protein, 3 μl 10× E1 enzyme, 3 μl 10× E2 enzyme, 3 μl 10× MuRF1 enzyme or rTcpC, 3 μl 10× Mg²⁺-ATP solution. Reactions were incubated at 37°C for 1 h and the products were subjected to Western Blot analysis [19]. To determine the MyD88-specific E3 activity of rTcpC, E3 and its substrate S5a in the kit were replaced by rTcpC and lysates of macrophages or rMyD88 (Abcam, USA), respectively.