Fig. 2: Interaction studies of the p632/p732 hetero-tetramer and the two homo-tetramers with the selected DARPins using Isothermal Titration Calorimetry (ITC).
The experiments show a specific and very high-affinity binding of the DARPin to only the p632/p732 hetero-tetramer. A ITC data for DARPin F11. B ITC data for DARPin A2. The top diagram displays the raw measurement and the bottom diagram shows the integrated heat per titration step. The KD value for the interaction is given. Thermodynamic parameters are provided in Supplementary Fig. 2B. C Pulldown experiments with the DARPins F11, A2 and a control DARPin. H1299 cells were transiently transfected with the indicated expression plasmids. The input signals are shown on the left, the signals after pulldown on the right. The p73 E363K and p63 K377E mutants enforce hetero-tetramer formation, the p73 E363R, K370E, E373R and R390D (EKER) mutant prevents it. D Pulldown experiments with the DARPin F11 and HaCaT cells. The left side shows the input signals, the right side the signals following pull-down experiments with the DARPin. The upper panel represents Western blot results with an anti-p63 antibody, the lower panel the corresponding results with an anti-p73 antibody. The time-line indicates the time following treatment with calcium, which results in a transient spike of p73 expression.
