Immunoblotting of EGFR and Cell Cycle Proteins

Cells treated with 8PN and/or EGFR TKI (afatinib or AZD9291) were lysed in NETN lysis buffer (150 mm NaCl, 1 mm EDTA pH 8.0, 20 mm Tris–HCl pH 8.0, 0.5% NP40) after 24 h. Immunoblotting was conducted as previously described [6 (link)], except that we used total of 10% sodium dodecyl sulfate–polyacrylamide gels for protein separation. After 1 h of blocking, primary antibodies were used against the following targets: CDCP1 (ab1377; Abcam, Cambridge, MA, USA), retinoblastoma protein (RB; #2947; Cell Signaling Technology, Danvers, MA, USA), phosphorylated RB (#8516; Cell Signaling Technology), cyclin E1 (ab33911; Abcam), cyclin E2 (ab40890; Abcam), EGFR (sc‐373746; Santa Cruz, Dallas, Texas, USA), phosphorylated EGFR (p‐EGFR; ab1377 and ab32894; Abcam), phosphorylated ERK (p‐ERK; #9101; Cell Signaling Technology), ERK (#9102; Cell Signaling Technology), and caspase 3 (#9661s; Cell Signaling Technology, or ab1899; Millipore, Billerica, MA, USA). GAPDH (10494‐1‐AP; ProteinTech, Rosemont, IL, USA), and EF1α (#05‐235; Millipore) were used as internal controls. Blotted proteins were detected using an enhanced chemiluminescence system (Millipore) with the BioSpectrum Imaging System (UVP, Upland, CA, USA).