1. DNA damage induces complex signal activation dynamics across many signaling pathways.
DNA damage induces complex signal activation dynamics across many signaling pathways. (A) Representative quantitative immunoblotting assays, shown for phospho‐ERK and phospho‐JNK. (a, b, d, e) Assay validation and linearity. Cells were starved and serum stimulated to activate ERK, or UV irradiated (50 J/m2) to activate JNK. Lysates from these positive controls (pc) corresponding to 2.5–30 μg of total protein from these positive controls (pc), along with 10 μg of total protein from untreated negative control (nc) cells were analyzed by western blotting for active ERK and JNK using phospho‐specific antibodies. Quantified band intensity was plotted against protein loaded to verify signal linearity. Gray diamonds indicate the signal intensity obtained from 10 μg of negative control lysate, and indicate high signal to noise ratio. (c, f) Representative western blots for phosphorylated ERK and JNK from lysates (10 μg total protein/lane) at the indicated time points following each of the six treatment conditions. Positive and negative control (pc and nc) lysates (10 μg/lane) were included in all gels as internal controls for normalization. (B) Quantification of western blot data for all signaling measurements of activity under the six treatment conditions were investigated. Measured signals quantified include p‐ATM (S1981), p‐Nbs1 (S343), p‐H2AX (S139), p‐p53 (S15), p‐p38 (T180/Y182), p‐JNK (T183/Y185), p‐Akt (S473), p‐ERK1/2 (T202/Y204), and total p53 and quantification of ELISA data for activation status of NF‐κB. Data are the mean of two to three independent experimental replicates and normalized to the signal measured at 0 h (prior to treatment) to yield a measure of ‘Signal Intensity.’ Source data is available for this figure in the Supplementary Information.
Andrea R Tentner et al. Mol Syst Biol 2012;8:568
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