The Journal of Neuroscience, September 22, 2004, ():
Cycling of NMDA Receptors during Trafficking in Neurons before Synapse Formation
J. Neurosci. Washbourne et al.
24: 8253
Supplemental data
Files in this Data Supplement:
- supplemental material
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Figure 1: Synthesis of anti-GFP Fab fragment
Fab fragment was generated by papain cleavage of anti-GFP monoclonal
antibodies and labeled with Alexa 568. SDS gel electrophoresis shows that
the Fab fragment was labeled by viewing under UV (A) and pure by staining
the same gel with Coomassie blue (B) or staining a Western blot with
anti-mouse secondary antibody (C).
- supplemental material
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Figure 2: The R1JHL antibody recognizes NMDARs at synapses
8 DIV cortical neurons were fixed, permeabilized with saponin and stained
for NR1 (using the R1JHL antibody), NR2A, NR2B and the vesicular glutamate
transporter 1 (VGlut1). Colocalization of the presynaptic marker, VGlut1,
and all NMDAR subunits is evident at synapses (arrows). Scale bar = 10mm.
- supplemental material
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Figure 3: Limited overexpression and dendritic localization of EGFP-NR1
4 DIV EGFP-NR1 transfected neurons were fixed and stained for NR1 (A) and
MAP2 (B) to show levels of expression of NR1 in dendrites of transfected
versus neighboring non-transfected cells. EGFP-NR1 was not present in the
axon since all GFP immunofluorescence colocalizes with MAP2, a protein
found exclusively in dendrites. Moreover, levels of overexpression of
EGFP-NR1 was limited as determined by comparing the number of clusters per
length of dendrite and the average intensity of NR1 clusters in transfected
versus neighboring non-transfected neurons from the same image (see
Washbourne et al.2002a for quantification and methods).
- supplemental material
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Movie 1. Movement of surface-labeled EGFP-NR1
An EGFP-NR1 transfected cortical neuron was imaged every 20 sec after
surface-labeling with anti-GFP antibody. Total imaging period is 4 min 20
sec. Scale bar = 15 mm.
