Analysis of de novo protein synthesis

This assay was performed as described^{18 (link), 20 (link)}. Briefly, ST cells were washed with PBS and cultured with methionine-free DMEM (GIBCO-Life Technologies) at 37 °C for 1 h prior to the addition of 1 μM tylophorine and 50 μM Click-iT^® HPG (L-homopropargylglycine [a reactive methionine analog], Invitrogen) for another 1 h and followed by infection of TGEV at an MOI of 7 for 1 or 3 h. The cells were then harvested and subjected to the ‘click’ reaction with 20 µM TAMRA (tetramethylrhodamine, Invitrogen) for labelling the de novo synthesized proteins in Click-iT® Protein Reaction Buffer according to the manufacturer’s protocol (Invitrogen). The resultant cell lysates were immunoprecipitated with anti-IκBα (Cell Signaling Technology) overnight at 4 °C with constant agitation prior to incubation with protein G agarose (Millipore) at 4 °C for another 2 h. After three to five washes, the specific immunoprecipitated protein was eluted and analyzed by western immunoblot analysis with the antibody against TAMRA (Thermo Fisher Scientific).