2.11. Cellular thermal shift assay (CETSA)

Engagement between FGFR1 protein and GZD824 was assessed by CETSA. For each group, 2 × 10⁷ cells were collected and resuspended in PBS after treatment with GZD824 (1 μM for H1581, 100 nM for Ba/F3‐FGFR1‐V561F) or DMSO 4 h, then the cell suspensions of each group were divided into eight equal parts. Pairs consisting of one experimental aliquot and one DMSO aliquot were heated from 38.0 to 57.8℃ for 3 min. Then, the samples were exposed to liquid nitrogen for 10 min, and then placed in a water bath at 37℃ for 10 min. After three freeze–thaw cycles, cell lysates were centrifuged at 12000 rpm at 4℃ for 10 min, and then boiled for 15 min. The soluble supernatant was used for WB analysis.