Radiolabeling of Huh7 Cells Expressing tRH Variants

Huh7 cells were seeded onto the wells of 6-well cell culture plates at a density of 2 × 10⁵ cells/well and incubated overnight to allow cell attachment. Cells were transfected with 50 nM and 100 nM of 5′ tRH^Gly (5′-GCAUUGGUGGUUCAGUGGUAGAAUUCUCGCCU-3′), 5′ tRH^Val (5′-GUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCU-3′), or scramble (5′-GCAUUCACUUGGAUAGUAAAUCCAAGCUGAA-3′)21 (link) (all from Integrated DNA Technologies, Coralville, IA) oligonucleotide after replacing cell culture medium with methionine- and cysteine-deficient DMEM (Life Technologies) and cultured for further 12 hrs. Cells were then metabolically radiolabeled for 12 hrs with 200 μCi/well of Express Protein Labeling Mix containing [³⁵S]methionine and [³⁵S]cysteine (PerkinElmer, Waltham, MA) in the presence or absence of 50 μg/ml puromycin and lysed with lysis buffer (20 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 1% Triton X-100, 0.05% SDS, and 10% glycerol) supplemented with 50 mM NaF, 5 mM Na₃VO₄, and a protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). The protein concentration of cell lysates was determined by the Bio-Rad Protein Assay (Bio-Rad), and 10 μg (total protein) of cell lysates was subjected to SDS-PAGE followed by staining gels with the Sypro Ruby Protein Gel Stain (Bio-Rad, Hercules, CA) and autoradiography.