Western Blot Analysis of Signaling Proteins

Protein extracts were run on an 8% SDS-PAGE gel, transferred to Immobilon-P transfer membranes (Millipore Corp., Bedford, MA), and visualized with antibodies (Abs) specific for GAB1 (Cell Signaling), p-MEK (Ser217/221; Cell Signaling), total MEK (Cell Signaling), p-AKT (Thr 308; Cell Signaling), total AKT (Cell Signaling), IE86 (monoclonal antibody [MAb] 810; Millipore), UL138 (14 (link)), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Abcam). Relative levels of intensity of bands detected by Western blotting were quantitated using ImageJ software. In each case, the relative intensity of each band was compared to that of the control protein (total MEK or total AKT [Fig. 2] or GAPDH [Fig. 1, 3, and 4]). The ratio of sample to control was set to a value of 1 for the mock treatment (Mock) or the first time point, and each subsequent sample/control ratio is presented as a multiplier of the reference time point.