The SW480 cells (have been incubated with BBR for 30 min) were collected, washed three times with cold PBS, and then lysed in PBS supplemented with protease inhibitors following three cycles of freeze–thaw. Cell lysates were then centrifuged at 13,000 rpm for 15 min, the supernatant was diluted with cold PBS and divided into two equal parts, with one part treated with DMSO and the other part treated with BBR (100 μM). After incubation at room temperature for 30 min, the two parts were divided into nine aliquots (60 μL) respectively, then heated at temperatures of 40, 44, 48, 52, 56, 60, 64, 68 and 72 °C for 3 min. The samples were cooled for 3 min at room temperature and kept on ice. Then, every aliquot was centrifuged and the supernate was analysed by Western blot (Lv et al. 2018).

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