H actin

Protein samples were separated by SDS–PAGE and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After blocking, the membranes were incubated with specific antibodies against different proteins at room temperature for 2 h or 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was visualized by enhanced chemilumines-cence (GE Healthcare, Piscataway, NJ, USA). Antibodies were against NF90 (interleukin enhancer binding factor 3, BD Biosciences, Franklin Lakes, NJ, USA; Epitomics, Burlingame, CA, USA), cyclin E1 (Epitomics), cyclin D1 (Epitomics), Myc (Sigma, St Louis, MO, USA), h-actin (Sigma) and GAPDH (Sigma).

Western immune-assessment of wild type and vIII variant of EGFR was performed as per previously described protocols [26 (link), 30 (link)]. Briefly, Antibodies used in this study for the detection of total EGFR, and phospho-EGFR (Y1080), were obtained from Cell Signaling; antibody for detection of h-actin was obtained from Sigma (St. Louis, MO).