![Figure 2. Association of EphA3 and EphB2 in intact cells. (A) Colocalization of AP-EphA3 and GFP-EphB2 in cells. HEK293 cells expressing biotinylated AP-EphA3 were transfected with GFP-EphB2. After 24 h, cells were stained with Alexa Fluor 647 α-EphA3 (IIIA4; red) before (top) or after (bottom) the addition of Alexa Fluor 594 SA–coated beads for 20 min, fixed, and imaged by confocal microscopy. (B) Cointernalization of EphA3 and EphB2. HEK293 cells were transfected with Wt diHcRed-EphA3 and Wt GFP-EphB2 or inactive mutant GFP-EphB2[3YF]. After 24 h, cells were stimulated for 20 min with cross-linked Alexa Fluor 647 IIIA4 before fixation and confocal microscopy. (A and B) Arrows indicate prominent colocalization. Insets show details in boxed areas at higher magnification. (C) Fluorescence lifetime microscopy shows close association of YFP-EphB2 and Wt or diHcRed-EphA3[3YF] in live cells. COS7 cells were transfected with YFP-EphB2 and Wt or 3YF mutant diHcRed-EphA3, and FRET between YFP and diHcRed was analyzed by FLIM before and after 40 min of stimulation with clustered IIIA4. YFP fluorescence lifetime maps are illustrated together with confocal micrographs of YFP-EphB2 and diHcRed-EphA3–transfected COS7 cells. All scale bars are in micrometers.](https://cdn.rupress.org/rup/content_public/journal/jcb/195/6/10.1083_jcb.201104037/8/jcb_201104037r_rgb_fig2.jpeg?Expires=1771715766&Signature=CpsTL75f~DAaKrbqvzqWPdlqog7dJqppv9nT9cDmhPJlBufLdQIdSwPSs6qmfXcXLyBnX3zVWgrRPUq9RUONatH2wpvjU9KtfTRPL8iBe3opmFJ89TM7YIi1XdEDxcZ9MyPW1ko~Utx8sq0hin80~JMpHEt8~RGBqN1vX~p2TRqer1S5oahfwikHVIY6f7VpEwwidoTTbZCxXtL3mTQC2m~6UUc9hnWP1zTdz~O2EWYbiZnY6zLPcgo5sktHwpvHBx8jpBP1csES~t30L3nipUDBUgxB~TrHtquCGiuJsoTFgW-tGTzlQbHN0kDFVpUCsqYO9u-KkmHG8k53nzkTnw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Association of EphA3 and EphB2 in intact cells. (A) Colocalization of AP-EphA3 and GFP-EphB2 in cells. HEK293 cells expressing biotinylated AP-EphA3 were transfected with GFP-EphB2. After 24 h, cells were stained with Alexa Fluor 647 α-EphA3 (IIIA4; red) before (top) or after (bottom) the addition of Alexa Fluor 594 SA–coated beads for 20 min, fixed, and imaged by confocal microscopy. (B) Cointernalization of EphA3 and EphB2. HEK293 cells were transfected with Wt diHcRed-EphA3 and Wt GFP-EphB2 or inactive mutant GFP-EphB2[3YF]. After 24 h, cells were stimulated for 20 min with cross-linked Alexa Fluor 647 IIIA4 before fixation and confocal microscopy. (A and B) Arrows indicate prominent colocalization. Insets show details in boxed areas at higher magnification. (C) Fluorescence lifetime microscopy shows close association of YFP-EphB2 and Wt or diHcRed-EphA3[3YF] in live cells. COS7 cells were transfected with YFP-EphB2 and Wt or 3YF mutant diHcRed-EphA3, and FRET between YFP and diHcRed was analyzed by FLIM before and after 40 min of stimulation with clustered IIIA4. YFP fluorescence lifetime maps are illustrated together with confocal micrographs of YFP-EphB2 and diHcRed-EphA3–transfected COS7 cells. All scale bars are in micrometers.
