Figure 1.
The PDZ binding domain of EphB2 is required for EphB colocalization with AMPA receptors but not NMDA receptors. a, Representative image of 7 DIV rat cortical neuron transfected with fEphB2 at 4 DIV and immunostained for FLAG epitope (red) and NR2B (green). Yellow indicates colocalization. b, Representative image of 7 DIV rat cortical neuron transfected with fEphB2ΔPDZ at 4DIV and immunostained for FLAG (red) and NR2B (green). Yellow indicates colocalization. Scale bar, 5 μm. c, Quantification of percentage of FLAG-positive puncta colocalized with GluR2,3 puncta in dendrites of neurons transfected with fEphB2 (n = 35), fEphB2ΔPDZ (n = 30), or fEphB2_KD (n = 45). d, Representative image of 7 DIV rat cortical neuron transfected with fEphB2 at 4 DIV and immunostained for FLAG (red) and GluR2,3 (green). Yellow indicates colocalization. e, High-magnification view of boxed region of dendrite from fEphB2-transfected cell. Arrows and yellow puncta indicate colocalization. f, Representative image of 7 DIV rat cortical neuron transfected with fEphB2ΔPDZ at 4 DIV and immunostained for FLAG (red) and GluR2,3 (green). g, High-magnification view of boxed region of dendrite from fEphB2ΔPDZ-transfected cell. Arrows indicate adjacent but non-overlapping puncta of GluR2,3 (green) and fEphB2ΔPDZ (red). h, Representative image of 7 DIV rat cortical neuron transfected with fEphB2_KD at 4 DIV and immunostained for FLAG (red) and GluR2,3 (green). In merged image, yellow indicates colocalization. Scale bar, 20 μm. i, High-magnification view of boxed region of dendrite from fEphB2_KD-transfected cell. Arrows and yellow puncta indicate colocalization. Scale bar, 2 μm. j, Quantification of GluR2,3 puncta density in dendrites of neurons transfected with GFP alone (n = 12) or GFP plus fEphB2, fEphB2ΔPDZ, or fEphB2_KD. k, Representative dendrites from 7 DIV rat cortical neurons cotransfected at 4 DIV with GFP and fEphB2, fEphB2ΔPDZ, or fEphB2_KD. Scale bar, 2 μm. l, Quantification of dendritic spine density in 7 DIV neurons transfected with fEphB2 (n = 18), fEphB2ΔPDZ (n = 16), or fEphB2_KD (n = 27). Error bars indicate SEM. *p < 0.005; **p < 0.0001.