35s methionine

Cells were labeled for 15, 30, 45 or 60 minutes at 37°C in methionine-free DMEM medium containing either 150 μCi/ml [³⁵S] methionine (PerkinElmer Life Sciences, Boston, MA) (for cytoplasmic protein labeling) or 150 μCi/ml [³⁵S] methionine and 100 μg/ml emetine (for mitochondrial protein labeling) as described (Leary and Sasarman, 2009 (link)).

HeLa cells were used for cellular metabolic labeling experiments. HeLa cells (20,000 per well) were seeded in 96-well plates. Twenty-four hours after seeding, drugs were added at the indicated concentrations, followed by addition of an aliquot of 1 μCi of [³H]-thymidine, [³H]-uridine or [³⁵S]-methionine (Perkin Elmer) for 1 h. Cells were washed twice with 200 μl of cold PBS and lysed by addition of 100 μl lysis buffer [20 mM Tris-HCl, pH 7.4, 1% SDS, 100 μg/ml yeast tRNA (for [³H]-thymidine incorporation and [³H]-uridine incorporation) or 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 100 μg/ml BSA (for [³⁵S]-methionine incorporation)]. After a 20-minute incubation at 4 ° C, 100 μl of 20% trichloroacetic acid (TCA) was added and the resulting mixtures were transferred to Millipore MSFCN6B10 filter plates. After 2 hours incubation at 4 ° C, t he mixtures were filtered under vacuum, and the plates were washed twice with 100 μl 5% TCA and twice with 100 μl ethanol. The plates were dried overnight, and 50 μl of Optiphase Supermix was added to each well and incorporation of ³H or ³⁵S was quantified by scintillation counting on 1450 Microbeta JET instrument (Perkin Elmer).

Cells were infected at an MOI of 10 with WR or vA3i and incubated at 37°C in the presence or absence of IPTG. At each time point, cells were labeled with culture medium without methionine containing 20 μCi/ml of [³⁵S]methionine (Perkin Elmer). After 30 minutes incubation at 37°C, the media was removed and the cells were harvested in sample buffer. Proteins were analyzed by SDS-PAGE followed by autoradiography (Condit & Motyczka, 1981 (link)).
For pulse-chase experiments, infected cells were pulse-labeled after 8 hours of infection with 10 μCi/mL of [³⁵S]methionine (Perkin Elmer) for 30 minutes. Some cells were harvested in sample buffer immediately after the pulse. For the remaining cells, the label was washed out and the cells were incubated at 37°C for varying times in the presence or absence of IPTG and absence of [³⁵S]methionine. Proteins were analyzed by SDS-PAGE followed by autoradiography.