Horseradish peroxidase conjugated anti mouse or anti rabbit immunoglobulins

Whole-cell protein extracts were used for analysis, and immunoblotting was conducted as described previously ^{22 (link)}. Antibodies against CDK4 (clone D9G3E; Cell Signaling Technology, Danvers, MA), CyclinD1 (clone G124-326; BD Biosciences, Franklin Lakes, NJ), GAPDH (clone 6C5, Ambion), p16INK4a (clone G175-405, BD Biosciences) were used as probes, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins (Jackson Immunoresearch Laboratories, West Grove, PA) were employed as secondary antibodies. The LAS3000 charge-coupled device (CCD) imaging system (Fujifilm Co. Ltd., Tokyo, Japan) was employed for detection of proteins visualized by Lumi-light Plus Western blotting substrate (Roche, Basel, Switzerland).

Whole-cell protein extracts were used for analysis, and immunoblotting was conducted as described previously [22 (link)]. Antibodies against E-cadherin (Mouse MAb IgG2a, 610181, BD Transduction Lab, USA), p53 (MAb clone DO-1; IgG2a; OP43-100UG, Calbiochem, Merck KGaA, Darmstadt, Germany), Cyclin D1 (MAb clone G124-326; IgG1; 554180, BD Pharmingen, USA), Vinculin (mouse IgG1; V9264, Sigma-Aldrich, USA), MCM7 (Mouse MAb IgG1; sc-9966, Santa Cruz Biotechnology, USA), CDK4 (Rabbit Pab; Cell Signalling; 610147, BD Transduction Laboratories, USA) were used as probes, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins (Jackson Immunoresearch Laboratories, PA, USA) were employed as secondary antibodies. The LAS3000 charge-coupled device (CCD) imaging system (Fujifilm Co. Ltd., Tokyo, Japan) was employed for the detection of proteins visualized by Lumi-light Plus Western blotting substrate (Roche, Basel, Switzerland).

The expression levels of EGFR, p-EGFR, PI3K, AKT, p-AKT, MAPK, and p-MAPK as well as vinculin were examined by western blotting. The western blot was performed as described previously^{37 (link)}. To expose one blot to several antibodies at the same time, the membrane was cut into several pieces at desired positions prior to exposure to specific antibodies. Antibodies against EGFR (Cell Signaling Technology, USA, Cat# 2646), p-EGFR (Cell Signaling Technology, USA, Cat# 2234), PI3K (Cell Signaling Technology, USA, Cat# 4249,), AKT (Cell Signaling Technology, USA, Cat# 9272S), p-AKT (Cell Signaling Technology, USA, Cat#9271S), MAPK (Cell Signaling Technology, USA, Cat# 9102,), p-MAPK (Cell Signaling Technology, USA, Cat# 9101) and vinculin (Sigma-Aldrich, USA, Cat# V9264) were used as primary antibodies. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins (Jackson Immunoresearch Laboratories, West Grove, PA) were used as secondary antibodies.