A549 and H460 cells were harvested and washed twice with cold PBS (Invitrogen). A radioimmunoprecipitation assay lysis buffer (Invitrogen) was used to solubilize the cells according to the product protocol, and then, the cell lysates were centrifuged at 12 000 g and 4°C for 10 min to obtain the supernatant. A DC protein assay kit (Bio‐RAD) was used to determine the concentration of proteins in the supernatant. The protein samples were then boiled in the loading buffer to obtain heat‐denatured protein, which was then separated using 5% SDS–PAGE (polyacrylamide gel electrophoresis) and transferred to an Immobilon‐P membrane (Millipore, Bedford, MA) for 2 hours (120 V). PBS containing 0.1% Tween 20 and 5% non‐fat dry milk was used to incubate the membrane for 1 hour to block non‐specific binding. The primary antibody (anti‐EGFR antibody, 1:1000, SCBT, Santa Cruz, CA) was used to incubate the membrane at room temperature for 2 h, and the membrane was then washed twice with PBS containing 0.1% Tween 20. In the next step, a secondary antibody (1:10 000, SCBT, Santa Cruz, CA) was used to treat the membrane for 60 min, and the membrane was then washed twice with PBS containing 0.1% Tween 20. Enhanced chemiluminescence detection reagents (Amersham Biosciences) were used to detect the bound antibody in accordance with the manufacturer's protocol. Each experiment was performed at least three times.

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