The Journal of Neuroscience, January 18, 2006, ():
Essential Contribution of the Ligand-Binding 
B/C Loop of PDZ1 and PDZ2 in the Regulation of Postsynaptic Clustering, Scaffolding, and Localization of Postsynaptic Density-95
J. Neurosci. Nonaka et al.
26: 763
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. GST-pull down assay to monitor the binding affinity of PDZ1mΔ and PDZ2mΔ towards CRIPT, a specific PDZ3 ligand.
Mutated PDZ domains, PDZ1mΔ and PDZ2mΔ, failed to bind to a specific PDZ3 ligand, CRIPT, even though the designed mutations still retained apparent sequence homology with the PDZ3 domain structure. Consistent with a previous report, the N326S mutation in the PDZ3 domain indeed reduced its binding affinity to CRIPT. GST (glutathione-S-transferase)-fused PDZ domains were expressed in E. coli, purified using GT (glutathione)-Sepharose, and mixed with COS cell lysates expressing the GFP-CRIPT protein. After extensive washing, the captured GFP-CRIPT proteins were eluted by boiling, subjected to SDS-PAGE and immunoblotted with a mouse monoclonal anti-GFP antibody (upper panel). The amounts of the GST-PDZ domain proteins were similar, as confirmed by an immunoblot with a goat anti-GST antibody (lower panel). The binding of CRIPT to GST-PDZ3 was mediated by the PDZ-binding motif in CRIPT in this assay, as this binding disappeared when the pull down assay was conducted using the mutant CRIPT, CRIPT(V101A), in which the C-terminal PDZ-binding motif was mutated.
- supplemental material
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Supplemental Figure 2. The full-length proteins are predominant in neurons overexpressing the various PSD-95 constructs.
The integrity of the wild-type and various mutant PSD-95-GFP proteins overexpressed in cultured neurons was tested by Western blot analysis using an anti-GFP antibody. The full-length proteins constituted the predominant species, thus indicating that overexpression of the constructs did not result in substantial protein degradation. Rat hippocampal neurons were transfected with the wild-type or mutant PSD-95-GFP by electroporation (Amaxa Biosystems, Gaithersburg, MD), and were cultured in Neurobasal medium (Invitrogen) containing B-27 supplement and 0.5 mM L-glutamine for 12 days in 60 mm dishes. The cells were lysed in RIPA buffer, and 1/24, 1/8, and 3/8 volume portions of the total lysates were subjected to SDS-PAGE and immunoblotted with a monoclonal anti-GFP antibody. The full-length PSD-95-GFP proteins are approximately 120 kDa, as expected.
