Fig. 6

Pbx interaction domain controls En paracrine activity. (A) Phenotypic analysis of hexapeptide-mutated forms of En2 protein. Compared to their wild-type counterparts, the eye phenotype induced by the activation of hexapeptide mutants was drastically reduced. The error bars represent statistical errors. (B-D) Intercellular transfer of En2 hexapeptide mutant ex vivo. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24h and cell surface accumulation of the secreted protein was monitored by flow cytometry (B). Internalisation of fluorescein-labelled En2, or En2 WW>KK (green) was visualised (C) and quantified (D) after 1h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. (E,F) Internalisation of fluorescein-labelled En2, En2HD, Hexa-En2HD was visualised (E) and quantified (F) as described above. The error bars represent statistical errors (A) or the s.e.m. (B,D,F). **P<0.01, ***P<0.001.