Non reducing sample buffer

The cells were lysed 24 h after C-ion irradiation using cell lysis buffer (Cell Signaling Technology) for 30 min on ice and scraped using cell scraper (SPL Life Sciences, South Korea). The cell lysates were centrifuged at 12,000 g for 10 min at 4°C and the supernatants were collected. 200 μg of total protein from each sample was incubated overnight with the anti-FTS antibody at 4°C, followed by incubation with protein A/G agarose (Santa Cruz) for 1 h. Immunoprecipitates were washed twice for 5 min with cell lysis buffer at 4°C. Bead bound proteins were eluted with non-reducing sample buffer (Thermo Scientific) at 95°C for 3 min and then subjected to SDS-PAGE and western blot analyses.

For this procedure, 7.5 × 10⁶ HEK 293T cells were transfected with 10 µg/10 cm dish FLAG-STAT3 and 10 µg/10 cm dish HA-STAT3 plasmids for 24 h, followed by treatment with 20 µM 323–1 or 323–2 and 100 µM S3I-201 for 4 or 24 h Thereafter, 293T cells were collected in the medium and centrifuged at 800 g × 5 min and then washed with cold PBS twice before adding 500 μL Pierce™ IP lysis buffer (Thermo Fisher Scientific, MA, United States, cat. no. 87787), containing protease and phosphatase inhibitor cocktail without DTT (Roche, Basal, Switzerland, cat. no. 11836153001) into the cells. Cell lysates were passed several times through a 27^1⁄2-gauge needle to disrupt the nuclei. Then, 1–2 mg extracts were added with a pre-cleaned 50 µL slurry of Pierce™ DYKDDDDK magnetic agarose (Thermo Fisher Scientific, MA, United States, cat. no. A36797), and then, supernatants were collected with magnet and immunoprecipitated (IP) at RT on a rotator for 30 min. Beads were boiled at 95°C for 5 min followed by the addition of 100 μL of 1x non-reducing sample buffer (Thermo Fisher Scientific, MA, United States, cat. no. 39000) into beads. The supernatants were collected with a magnet and then added 5 μL of 1M DTT (Thermo Fisher Scientific, MA, United States, cat. no. P2325) into samples before proceeding with immunoblotting.