35S-methionine/cysteine pulse-chase assay

Radiolabel pulse-chase assays of Flp-In T-REx 293 cells transiently expressing the model ERAD substrate NHK-HA were performed as described previously (Christianson et al., 2008). Briefly, cells were starved in DMEM lacking methionine and cysteine (Lonza)+10% dialysed FBS for 10 min, metabolically labelled by supplementing starvation medium with ³⁵S-methionine/cysteine (EXPRE³⁵S³⁵S Protein Labelling Mix (PerkinElmer), 80 µCi/6 cm plate) for 15 min, rinsed three times in Dulbecco's PBS, and chased for indicated time points in DMEM supplemented with methionine and cysteine (50 mM each). Cells were lysed in buffer containing 1% Triton X-100 as above, and the detergent-soluble, post-nuclear lysates pre-cleared using mouse IgG conjugated to agarose beads followed by immunoprecipitation with anti-HA-agarose beads (Sigma). Bead-bound radiolabelled substrates were resuspended in Laemmli buffer+20 mM DTT, separated by SDS-PAGE and band intensities quantified by phosphorimager and QuantityOne software (Bio-Rad). After background subtraction, half-life (t_1/2) values were calculated (GraphPad Prism 7) using data from at least three independent experiments, from which the average and s.e.m. values for each time point were determined. Significance was determined by Students t-test.