Phosphorylated rb

Total protein was extracted from whole cells and 20 μg of isolated protein was separated by SDS-PAGE and electroblotted onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were then probed with antibodies against FOXO1, p21, CyclinD1, phosphorylated Rb, Rb, and α-Tubulin (Cell Signaling, Danvers, MA, USA), using standard protocols.

Cell pellets were lysed in RIPA lysis buffer (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Cell lysate protein (25 µg) was separated in 4%-20% SDS polyacrylamide gel lanes and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk or 5% bovine serum albumin in TBST and incubated with primary antibodies targeted against β-actin (#4970), p21 (#2947), phosphorylated histone H3 (#53348), phosphorylated Rb (#9301), or Rb (#9309) (all from Cell Signaling Technology). The membranes were then washed, incubated with secondary antirabbit (#7074) and antimouse (#7076) IgG antibodies tagged with horseradish peroxidase and developed using SignalFire ECL Reagent or SignalFire Plus ECL Reagent (all from Cell Signaling Technology). All samples were probed with different antibodies on the same membrane, always including β-actin loading control. Membranes were either cut, or stripped of antibodies using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific). Densitometry was assessed using the ImageJ gel lane area calculation tool (ImageJ version 1.50i; https://imagej.nih.gov/ij).

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).