The EGFR phosphorylation sites showed distinct phosphorylation patterns after EGF stimulation in EGFR-WT and EGFR-mutant cells.

<p>H1299 cells stably expressed the empty vector, EGFR-WT or mutant EGFR-L858R. (A) After stimulation with EGF (10 ng/ml) in serum-free medium for 10 minutes, cell extracts were subjected to immunoblotting analysis with the indicated phospho-tyrosine antibodies. EGF-dependent (EGFR-Tyr992, -Tyr1148 and -Tyr1068) and EGF-independent [EGFR-Tyr845, -Tyr974, -Tyr1086 and -Tyr1101, -Thr654 (see below) and -Ser1046 (see below)] phosphorylation were observed in H1299 cells expressing EGFR-L858R mutant. The phosphorylation of EGFR-Thr654 (B) and -Ser1046 (C) with or without EGF treatment in different lung cancer cell lines was monitored via <i>in situ</i> PLA. The quantification of <i>in situ</i> PLA is shown on the right. EGFR-Thr654 and -Ser1046 were phosphorylated upon EGF treatment in H1299-EGFR-WT cells, whereas their phosphorylation was EGF-independent in H1299-EGFR-L858R cells. (D) Immunoblotting analysis of EGFR-Thr654 and -Ser1046 was performed in EGFR mutant cells (H1975, which contains the EGFR-L858R and -T790M mutants) and cells expressing EGFR-WT (A549). The phosphorylation of EGFR-Tyr1068 was induced by EGF. The phosphorylation of EGFR-Thr654 and -Ser1046 was EGF-independent in H1975 and H1299-EGFR-L858R cells as determined by immunoblotting.