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. 2015 May 15;142(10):1840–1849. doi: 10.1242/dev.114181

Fig. 6.

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 24 h 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 1 h 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.