Figure 5

Reverse phase protein arrays. Fibroblast protein lysates of the indicated genotypes (H-ras^-/-, N-ras^-/-, H-ras^-/-/N-ras^-/- and their WT control counterparts) were printed as indicated in Materials and methods onto slides containing two sets of spots corresponding, respectively, to WT controls and knockout samples after being subjected to the specified culture conditions (starvation or stimulation with serum for 1 hour or 8 hours). All samples were printed in duplicate, using five-point, two-fold dilution curves (starting at 2 μg/μl). The sixth point was always a negative control consisting of lysis buffer alone. After staining and development with the specific antibodies indicated on the y-axis, the slides were scanned and the ratios of the signals of the different ras knockout samples, normalized in relation to their respective WT controls, are depicted as bars on the x-axis of the graphs. Ratios smaller than 0.6 are considered to indicate decreased protein expression, whereas ratios higher than 1.5 are considered as indicative of increased expression. Similar results were obtained in two separate experiments. Results depicted here represent the proteomic validation of the genomic expression changes corresponding to (a) various apoptotic proteins, (b) proteins in the JAK/STAT signaling pathway, and (c) well known Ras effectors in fibroblasts.