Figure 2.

Picchu-X Screen to Identify Hits Affecting EGFR Activity

(A) The protein interaction sub-network of the 20 hits identified in a high-content siRNA screen and their connection to seed set proteins. The sub-network was extracted from a larger network of 533 proteins obtained from the Human Protein Reference Database (HPRD) based on their direct associations with seed-set proteins and 1 (or more) other network member (central rectangle indicates primary seed or EGFR; white ellipses indicate additional seeds; shaded polygons indicate screen hits).

(B) FLIM-intensity scatterplots and associated regression analysis of six example experimental groups (three non-hits and three hits). Each data point shows mean fluorescence lifetime plotted against mean fluorescence intensity for a single cell. Pearson correlation coefficients describe the association between lifetime and intensity for the EGF-treated (solid circles) and non-treated (empty circles) subgroups, and the difference in r values was used to evaluate the effects of target protein KD on EGFR activity (^∗p < 0.05).

(C and D) Lysates of the indicated EGF-stimulated HCC1954 cell lines were examined by western blotting. Bar chart in (C) summarizes densitometry analysis of phospho-EGFR levels normalized to tubulin (n = 3 independent experiments ± SEM (^∗p < 0.05, two-tailed Student’s t test). Similarly, immunoblots for phospho-CrkII are shown in (D).

See also Figures S1 and S2.