As further analysis, the concentrations of TAp63α and GAPDH were quantified by densitometry to produce a protein concentration-based normalization factor for the transcriptional activity of each clone. Normalizing this concentration-based normalization factor for transfection efficiency using the Renilla data led to the identification of the exactly same stretch of amino acids, albeit with different intensities as compared with Figures 1b and 2b. The activity data for the experimental series with both mutant libraries were re-processed with these normalization factors, thus removing a concentration-dependent influence on the measured activity (Figures 1b and 2b, Supplementary Figures S3 and S4). SAOS-2 cells were transfected in the same manner as for the transactivation assays with different p63-containing pCDNA3.1 plasmids (Effectene, Qiagen). Cells were harvested after 24 h, resuspended, and lysed in M-PER reagent (Pierce, Schwerte, Germany) for 5 min. After addition of 15 μl of 4 × SDS buffer containing 20% β-mercaptoethanol, samples were heated for 5 min at 95°C. A volume of 7 μl of the lysate was loaded onto a 17-well NuPage (Invitrogen, Karlsruhe, Germany) 4–12% Bis–Tris (SDS) polyacrylamide gel. Samples were transferred to a PVDF membrane (Immobilon-P 0.45 μM) (Millipore, Schwalbach, Germany) using an XCell II blot module. The blot was blocked in 5% skim milk and probed with mouse anti-myc antibody clone 4A6 (Millipore), anti-ubc-9 rabbit polyclonal antibody (Cell Signaling, Frankfurt, Germany), or anti-GAPDH (Chemicon International). Detection was performed using an HRP goat anti-mouse IgG peroxide conjugate (Sigma). The blots were quantitated using the Biometra BioDocAnalyze 2.0 software (Biometra, Göttingen, Germany). Each experiment was repeated three times.