Figure 2 | Scientific Reports

Figure 2

From: High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system

Figure 2

Autophosphorylation of EGFR-V5 induced by HA-EGF-FLO42 in yeast cells.

(a) EGF-dependent autophosphorylation of EGFR. Yeast cells co-expressing HA-EGF-FLO42 and EGFR-V5 were lysed and separated to cytoplasmic and cell wall fractions. Cytoplasmic fraction was subjected to western blot analysis using anti-V5 and anti-EGFR antibodies against phosphotyrosine-1068 (pY-1068), pY-1148 and pY-1173. Expression of HA-EGF-FLO42 in cell wall fraction was confirmed by using anti-HA antibody. Numbers shown at the bottom indicate lane number. Single and double asterisks indicate non-glycosylated and glycosylated forms, respectively. (b) Subcellular localization of HA-IL5-FLO42 in yeast cells. Intact cells were stained with anti-HA antibody. Bar = 2 μm. (c–e) Autophosphorylation of EGFR-V5 induced by HA-EGF-FLO42 in yeast cells. Spheroplasts were stained with a specific antibody against pY-1068 (c), pY-1148 (d) and pY-1173 (e) in the EGFR and then subjected to microscopic and flow cytometric analyses. HA-IL5-FLO42 was used as control of non-specific ligand for EGFR. Bars = 2 μm.

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