Pulse Chase Labeling of Proteins

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 At appropriate time points, aliquots were withdrawn and the reaction was stopped with cold PBS.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.