Fig. 6. EphB2–GluN1 interaction in HEK293T cells is charge-dependent.

a Representative images of PLA results in HEK293T cells. HEK293T cells were transfected with the indicated FLAG-EphB2 single point mutants (WT, Y504E, or Y504F), together with Myc-GluN1 WT, GluN2B, and EGFP. The upper panels show PLA signal alone. The lower panels are merged images of EGFP in green and PLA signal in magenta. Scale bar = 10 µm. b Quantification of the effects of EphB2 mutants on PLA puncta number. PLA puncta number are quantified by counting the number of puncta per 100 µm² in EGFP⁺ cells and normalizing to the WT condition. (****p < 0.0001, ANOVA; n = 30 cells for each condition). c Representative images of PLA results in HEK293T cells. HEK293T cells were transfected with the indicated Myc-GluN1 single point mutants, together with GluN2B, FLAG-EphB2 Y504E, and EGFP. The upper panels show PLA signal alone. The lower panels are merged images of EGFP in green and PLA signal in magenta. Scale bar = 10 µm. d Quantification of the effects of GluN1 mutants on PLA puncta number. PLA puncta number are quantified by counting the number of puncta per 100 µm² in EGFP⁺ cells and normalizing to the WT condition. (*p < 0.05, ***p < 0.005, ANOVA; n = 30 cells for each condition). e Representative images of PLA results in HEK293T cells. HEK293T cells were transfected with the indicated Myc-GluN1 double point mutants, together with GluN2B, FLAG-EphB2 Y504E, and EGFP. The upper panels show PLA signal alone. The lower panels are merged images of EGFP in green and PLA signal in magenta. Scale bar = 10 µm. f Quantification of the effects of GluN1 mutants on PLA puncta number (*p = 0.0219, ****p < 0.0001, ANOVA; green dots represent n = 30 cells for each condition).