Figure 1: Dynamic signalling responses to ErbB2 blockade.

(a) Biparatopic anti-ErbB2 DARPin (6L1G) inhibits phosphorylation of both ErbB2 and ErbB3 while trastuzumab (TZB) attenuates only p-ErbB3. ErbB2-overexpressing cell lines were treated with 100 nM of 6L1G or TZB; ErbB2 or ErbB3 was immunoprecipitated and total phosphotyrosine content (p-Tyr) was determined. Co-IP analysis of Grb2 binding to ErbB2 is also shown. (b) Sustained inhibition of both AKT and ERK signalling pathways after 6L1G treatment versus transient decrease of p-AKT after 6 h of TZB treatment and consecutive p-AKT rebound after 72 h. Cancer cell lines were treated as above and whole-cell extracts were analysed. (c–f) Dynamic signalling events induced by TZB and 6L1G: quantitative western blot time course analysis. Data are means±s.d. of integrated intensities (I.I., cf. Li-COR Odyssey manual), with 100% referring to I.I. of untreated cells (0 h). (c) Expression of ErbB2 and reduced phosphorylation at specific Tyr-residues (color-coded (Y877 to Y1248), *P value versus level at 0 h), (d) expression of ErbB3 and level of p-ErbB3 after 6L1G or TZB treatment (colour-coded significance levels; ErbB3 expression upregulation, *P value versus 0 h; p-ErbB3 Y1289 re-phosphorylation, *P value versus 30 min TZB) (e) expression of AKT and level of p-AKT (p-AKT re-phosphorylation, *P value versus 6 h TZB) (f) expression of ERK1/2 and level of p-ERK1/2 in BT474 and SKBR3 cells (p-ERK dephosphorylation, *P value versus 0 h). For representative blots see Supplementary Fig. 1A,B. (All statistical analyses are one-way ANOVA on ranks and post hoc Dunnett’s test.) (g–i) Inhibition of downstream signalling (g) and ensuing effects of proliferation (XTT assay; h) or induction of apoptosis (FACS; i) after 72 h of treatment with 1 μM of AKT/PKB inhibitor MK2206, 2 μM MEK1 inhibitor AZD6244 alone or in combination with 100 nM TZB or 6L1G (*P value versus MK2206 or as drawn by brackets in h, two-sided, unpaired Welch’s t-test; Data in i are mean±s.e.m., *P value versus ctrl or as drawn in i, two-sided, unpaired Welch’s t-test). Similar results were obtained with the AKT inhibitor GDC-0068.