A11577

Cells were fixed with 4% paraformaldehyde (PFA) for 15 min and with 1% NP-40 for 20 min at 25°C, and then washed with PBS three times. Afterward, 5% normal goat serum was used for blocking at 37°C for 30 min. The cells were incubated with rabbit anti-EGFR primary antibody (A11577, 1:100; ABclonal) overnight at 4°C, followed by incubation with fluorophore-conjugated secondary antibodies (A5011, 1:50; ABclonal) for 1 h. The cell nuclei were stained with DAPI (Beyotime) and cells were packaged on cover glasses with antifade mounting medium. Imaging was performed either with a Leica Tcssp8 laser scanning confocal microscope (Leica, Wetzlar, Germany) or a Leica DM4B positive fluorescence microscope (Leica).

Lenvatinib (HY-10981), gefitinib (HY-50895), and copanlisib (HY-15346A) were purchased from MedChemExpress (Shanghai, China), and elacridar (S7772) was purchased from Selleck Chemicals (Shanghai, China). Stock solutions of 20 mM Lenvatinib, 100 mM elacridar, and 20 mM gefitinib were dissolved in 100% dimethyl sulfoxide (DMSO), and the stock solution of 10 mM copanlisib was dissolved in Milli-Q water. Antibodies against total epidermal growth factor receptor (EGFR; A11577, ABclonal), phospho-EGFR (AP0820, ABclonal), total PI3K (ab32089, Abcam), phospho-PI3K (4228, CST), total AKT (9272, CST), phospho-AKT (4060, CST), total MEK1/2 (A4868, ABclonal), phospho-MEK1/2 (AP0209, ABclonal), total ERK1/2 (4695, CST), phospho-ERK1/2 (4376, CST), caspase-3 (T40051, Abmart), Bcl-2-associated X (Bax; T40044, Abmart), multidrug resistance protein 1 (MDR1; 13978, CST), breast cancer resistance protein (BCRP; 130244, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 5174, CST) were used. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Beyotime Biotechnology (Shanghai, China). Alexa Fluor-conjugated goat anti-rabbit (647 nm) and goat anti-mouse (488 nm) antibodies were purchased from Invitrogen (Shanghai, China).