Ab1899

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).

The retrieved liver tissues were homogenized and lysed in an ice-cold protein lysis buffer and centrifuged at 13000 rpm for 15 minutes at 4°C. Protein concentrations of the lysates were determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The lysates (20 μg/lane) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gels and electroblotted onto a nitrocellulose membrane (HybondTM-C Extra, Amersham Biosciences Corp., Hong Kong, China). The membrane was blocked for 30 minutes at room temperature in 3% nonfat milk Tris-buffered saline with 0.05% Tween 20, and then incubated overnight at 4°C with the indicated primary antibodies against NF-κB (1 : 500, MAB3026, Chemicon, Millipore, Temecula, CA) or caspase-3 (1 : 200, AB1899, Millipore, Temecula, CA), and then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. After washing, the membrane was developed using chemiluminescent substrates (WBLUC0100, Millipore, Temecula, CA). Densitometric analysis of the gels was performed using Gel-Pro analyzer software, and the results were expressed relative to the density of β-actin (1 : 500, MAB1501, Chemicon, Millipore, Temecula, CA).