Immunoprecipitation and glutathionylation of STAT3

A549 cells were cultured in 10 cm² cell culture plates and treated with 1 mM diamide and 45 μM ALT in the presence or absence of 3 mM NAC for 4 h. Cells were collected and lysed in IP lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 0.1 mM PMSF, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and Leupeptin). 800 μg proteins from the clarified cell lysates were incubated overnight at 4 °C with rotation in the presence of STAT3 antibody (1:100 dilution). After incubation with sepharose A/G beads (Beyotime, Biotechnology) for another 1 h at 4 °C with rolling end-over-end, the immune complexes were collected, washed 3 times with cold lysis buffer and separated from the beads by boiling in LDS non-reducing sample buffer (ThermoFisher Scientific) for 5 min. The immunoprecipitated proteins were subjected to Western blotting for the detection of STAT3 and protein linked GSH (PSSG) using respective antibodies.