Figure 2

Proof of principle experiments for the determination of G1/S transition point in trastuzumab resistant HCC1954 cells. A. Western blots showing the expression and activation of key G1/S proteins. Cells were treated either only with Lipofectamine (MOCK) or with 20 nM CDK4 siRNA for 24 hours. Then, cells were synchronized for 24 hours and subsequently stimulated with 25 ng/ml EGF for 6, 12, 18 or 24 hours. Cell lysates were applied to immunoblotting. B. Reverse Phase Protein Array (RPPA) showing the phosphorylation state of pRB protein (Ser 807/811). The same lysates (from A) were applied to RPPA. The upper panel shows the read-out of antibody signal at near infra-red range for phospho-pRB antibody with four replicates. The lower panel shows the graphical representation of phospho-pRB antibody signal intensity for two different conditions and for four time points. Signals were normalized to 0 hour MOCK sample.