Express 35s protein labeling mix

The half-lives of transfected FLAG epitope-tagged circadian clock proteins (BMAL1, PER2 and CRY1) in the absence (as a control) and presence of siRNAs targeting COPS7B were measured by metabolic pulse chase protein labeling according to the protocol of Zhou et al. Briefly, 72 hr post-transfection, 100 mm plates of HEK293T cells were starved of amino acids for 1 hr and metabolically labeled for 30 min with 100 μCi of Express-35S protein-labeling mix (PerkinElmer Life Sciences, Schwerzenbach, CH) in 1 ml methionine- and cysteine-free DMEM medium supplemented with 10% dialysed FCS (‘the pulse’). Sunsequently, cells were washed to remove unbound radioactive amino acids, and incubated in complete medium (‘the chase’). At indicated time points (0, 2, 4, and 6 hr), the cells were disrupted in ice-cold RIPA lysis buffer (0.02M Tris/pH7.2, 0.15M NaCl, 1% Trion X-100, 1% Na-deoxycholate, 0.1% SDS), and 1000 μg of whole cell extract were immunoprecipitated with Monoclonal ANTI-FLAG M2 Antibody from Sigma (F3165). Proteins were subjected to 9% SDS-PAGE and the gels were dried for 2 hr at 80°C by Model 583 gel dryer (BIO-RAD, Hercules, CA). Labeled proteins were visualized by autoradiography and quantitative analysis of three independent experiments for each circadian clock protein was performed with ImageStudio software.

Pulse chase experiments were used to monitor secretion of the bulk flow marker SP-FLAG-Cp and intracellular transport of Gas1, CPY, and the misfolded form of CPY, CPY*. These experiments were performed as previously described (Pagant et al., 2007 (link)). Briefly, strains were grown to mid-log phase at 30°C, starved for 15 min, and labeled for 5 min with 1 µl per OD of cells of EXPRESS ³⁵S Protein Labeling Mix (PerkinElmer) for 5 or 10 min. The label was chased with excess rich media and two OD aliquots of cells harvested at different times. Cells were lysed in detergent, and the protein of interest was immunoprecipitated from cell lysates, and cell media (when measuring SP-FLAG-Cp secretion) were separated by SDS-PAGE and detected by phosphorimaging using a Typhoon scanner (GE Healthcare). The protein bands were quantified using Fiji, and the percentage of the mature or secreted band in each sample was plotted with Prism 7.0 (GraphPad Software).

Cells were starved for 30 min in cysteine/methionine-free DMEM and then radiolabeled in the same medium containing 22 μCi/ml EXPRESS³⁵S protein labeling mix (PerkinElmer Life Sciences). After 30 min of incubation at 37 °C, the radiolabel was removed, and cells were washed with PBS and incubated in complete DMEM for various lengths of time. Cells were washed with PBS before being lysed in buffer A. Cell debris was removed by centrifugation (10,000 × g for 10 min at 4 °C). The lysates were precleared by adding protein A-Sepharose (Generon) and incubated for 30 min at 4 °C. For immunoisolation, anti-PrxIV antibody was added to protein A-Sepharose. Samples were incubated for 16 h at 4 °C with end-over-end mixing. The Sepharose beads were pelleted by centrifugation (600 × g for 1 min) and washed three times with buffer A. Proteins were eluted with IEF sample buffer prior to loading onto a one-dimensional IEF gel. Gels were fixed, dried, and exposed to a phosphorimaging plate. Radioactivity was detected using a Fujifilm FLA-7000 PhosphorImager. Quantification of band intensities was carried out using ImageJ software.

HEK293T cells (500,000 cells/ml) were plated in 12-well flat bottom culture cluster dishes (Costar, Fisher Scientific) 24 hr before the experiment. IPA (1 μM), or a combination of IPA with GSK2606414 (TRC inc. Toronto Canada), were added to the cells culture for no longer than 2 hr. 15 min prior to lysing cells, 4 μCi of Express [³⁵S] protein-labeling mix (Perkin–Elmer) was added. Media were removed, and cells were immediately lysed in a buffer containing 25 mM Tris-HCl pH 8, 8 mM MgCl₂, 1 mM dithiothreitol (DTT), 1% Triton X-100, 15% glycerol, 10 mM leupeptin, 153 μM aprotenin, and 1 mM phenylmethanesulfonyl fluoride. Cells lysates were kept on ice for 30 min before high-speed centrifugation followed by denaturation at 95°C. The samples were resolved on an SDS-polyacrylamide gel, which were then stained with Coomassie blue to ascertain protein loading. Dry gels were exposed to a blank phosphorscreen and scanned using a Typhoon variable mode imager (GE Healthcare). The resulting autoradiograms were quantified using ImageQuant software (Molecular Dynamics).

Pulse‐chase experiments were used to monitor intracellular transport of Gas1 and CPY and to monitor Sec23 and Sec24 degradation. These experiments were performed as previously described.²³ Briefly, strains were grown to mid‐log phase at 30°C, starved for 15 minutes, and labeled for 5 minutes with 1 μL per OD of cells of EXPRESS ³⁵S Protein Labeling Mix (PerkinElmer) for 5 minutes. The label was chased with excess rich media and 2 OD aliquots of cells harvested at different times. Cells were lysed in detergent and the protein of interest was immunoprecipitated from cell lysates, separated by SDS‐PAGE, and detected by phosphorimaging using a Typhoon scanner (GE Healthcare). The protein bands were quantified using Fiji and the percentage of the mature band in each sample was plotted with Prism 7.0 (GraphPad Software).