Gel purification kit

Ba/F3-EGFR-del19/C797S, Ba/F3-EGFR-del19, and L858R/T790M/C797S cells (1 × 10⁶ cells/ml) were exposed to 100 µg/ml N-ethyl-N-nitrosourea (ENU) (Sigma) for 24 h. After ENU treatment, the cells were washed with PBS and cultured on D-10 for 24 h. The cells were seeded at a density of 1 × 10⁵ cells/well in 96-well plates for 24 h and cultured with brigatinib (300, 600, 800, or 1000 nM), a combination of brigatinib (300, 600, 800, or 1000 nM), 10 µg/ml panitumumab, or gefitinib (300, 600, or 1000 nM) for 3 weeks. Drug-resistant cells were picked and expanded. DNA extracts were prepared from these clones by lysing them with proteinase K buffer, and the entire kinase domain of EGFR was amplified using KOD plus neo (TOYOBO). The PCR products were purified using a gel purification kit (GE Healthcare) and analyzed using standard Sanger sequencing.