Cell lysis buffer

Cancer cells grown in 100 mm dishes overnight at a density of 1 × 10⁶ cells per dish were exposed to MG132 for 4 h, followed by treatment with 100 μM TMZ–POH for another 24 h. Then cells were collected and lysed with cold Cell Lysis Buffer (Beyotime, Beijing, China), and incubated with antibody against Ubiquitin or isotype IgG (2 μg, Proteintech, Wuhan, China) overnight at 4 °C with gentle rotation. 40 μl Protein A + G Agarose (Santa Cruz Biotechnology, Inc, Dallas, USA.) was added to per tubes and rotated at 4 °C for 3 h. Beads were precipitated by centrifugation at 16,000 × g for 30 s and washed five times with cold Cell Lysis Buffer. The pellets were re-suspended in 1 × SDS-PAGE Sample Loading Buffer (Beyotime, Beijing, China), and incubated at 100 °C for 5 min. The supernatants were used for western blot analysis using the antibody against MGMT.

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.