Fig. 4

Biochemical profiling of EGFR^G724S. a Homogeneous time-resolved fluorescence (HTRF) is used for IC₅₀-determination for EGFR^19del and EGFR^19del+G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC₅₀-values with HTRF for the three generations of EGFR TKIs against EGFR^19del+G724S. Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR^19del, EGFR^G724S or EGFR^19del+G724S) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control (n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR^19del, EGFR^G724S, or EGFR^19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR, bound to 4-anilinoquinazoline based TKI PD168393, is shown