35s methionine cysteine expressed protein mix

Pulse chase experiments were performed as previously described^{51 (link),52 (link)}. Briefly, cells were trypsinised and starved for 30 min in methionine/cysteine free DMEM at 37 °C before pulse labelling. Cells were labelled for 10 min at 37 °C with 10 mCi/ml [³⁵S]methionine/cysteine (expressed protein mix; PerkinElmer) and chased for the indicated time points. At appropriate time points, aliquots were withdrawn and the reaction was stopped with cold PBS. Cell pellets were lysed in Tris buffer containing TX-100 and pre-cleared with agarose beads for 1 h at 4 °C. Immunoprecipitations were performed for 3 h at 4 °C with gentle agitation. Samples were eluted by heating in reducing or non-reducing sample buffer as indicated, subjected to SDS-PAGE and visualised by autoradiography.

Pulse chase experiments were performed as previously described.¹³ (link)^,17 (link)^,37 (link) Briefly, either mock or virus-infected cells were grown in culture (50μM MG132 was added to the overnight culture medium where indicated), collected by trypsinization and starved for 30 minutes in methionine/ cysteine free DMEM at 37°C before pulse labelling. Cells were labelled for 10 minutes at 37°C with 10 mCi/ml [³⁵S]methionine/cysteine (expressed protein mix; PerkinElmer) and chased for the indicated time points. At appropriate time points, aliquots were withdrawn and the reaction was stopped with 500 μl cold PBS. Cell pellets were lysed in Tris buffer containing 0.5% NP-40 and pre-cleared with agarose beads for one hour at 4°C. Immunoprecipitations were performed for three hours at 4°C with gentle agitation. Samples were eluted by boiling in reducing sample buffer, subjected to SDS-PAGE and visualized by autoradiography.