Photofluorography

For the pulse-chase analysis, HEK293T cells were grown as described before. For the transient transfection, 3 × 10⁶ cells were plated in a 25 cm² flask and grown overnight. The following day, the cells were transfected using Lipofectamine Plus (Invitrogen), according to the manufacturer’s instructions. The total amounts of plasmid DNA in all samples were equalized with empty vector DNA as appropriate. After 24 h, the transfected HEK293T cells were harvested via scraping, washed with PBS, and suspended in 7 mL of labeling medium (methionine- and cysteine-free RPMI [MP Biomedical, Solon, OH] containing 5% fetal calf serum [FCS]). The samples were incubated for 20 min in either the presence of MG132 (10 μM) or the equivalent volume of DMSO at 37°C to deplete the intracellular methionine/cysteine pool. The cells were then labeled for 15 min at 37°C in 200 μL of labeling medium supplemented with 30 μL (300 μCi) of [³⁵S]-Expre³⁵S³⁵S-label (NEG072; PerkinElmer). After the labeling period, unincorporated isotope was removed, and equal aliquots of cells were added to 1 mL of prewarmed complete DMEM and chased for the selected times. Cells and virus-containing supernatants were harvested separately at each time point and were stored on dry ice until all samples had been collected. For the immunoprecipitation, the cells were lysed in 1 mL of Triton X-100-based lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol) and incubated at 4°C on a rotating platform for 20 min. After lysis, insoluble material was pelleted at 10,000 rpm for 10 min, and the clarified supernatants were added to protein A-Sepharose beads (Sigma-Aldrich, St. Louis, MO; cat. number P3391) that had been preadsorbed with anti-GSN polyclonal antibodies (5 μL plasma/IP in 1 mL lysis buffer). The samples were adjusted to a 1.3 mL total volume with lysis buffer and were incubated for 1 h at 4°C on a rotating platform. The beads were then washed three times with 1 mL each of lysis buffer. The precipitated proteins were solubilized via boiling in 100 μL SB, and they were separated via SDS-PAGE. The gels were fixed and dried. The gels were exposed to Kodak XMR film, and the proteins were visualized via fluorography. For protein quantitation, the gels were exposed to imaging plates. The plates were read using a Typhoon FLA 9500 Phosphoimager, and the quantitation of the data was done using FujiFilm Multi Gauge software.