Cellular thermal shift assay [24]

PC3 cells were washed in 1X PBS before splitting evenly at 1 × 10⁶ cells into 2-mL centrifuge tube in complete medium. One of the 2-mL centrifuge tubes were treated with 0.1% DMSO, while others were treated with indicated concentrations of FKB. After 2 h incubation at 37 °C in a water bath, the cells were washed and suspended in 600 μL of 1X cold PBS that containing protease inhibitor. Each of the treated cells were split evenly into nine new 2-mL centrifuge tubes, labeled with temperature ranging from 40 to 62 °C in increments of 3 degrees. The cells were incubated for 3 min at the indicated temperature, followed by 3 min incubation at room temperature before snap-frozen in liquid nitrogen. The cells were then lysed via snap-frozen in liquid nitrogen, thawing at 25 °C, and brief vortexing three times, before being spun down at 13,000 rpm for 15 min at 4 °C. Prior to heating at 75 °C for 10 min, 40 μL of the supernatants were mixed with 10 μL 5X loading dye containing β-mercaptoethanol, and then used for Western Blot analysis. Primary antibodies against the protein of interest were used.