The Journal of Neuroscience, February 22, 2006, ():
Transsynaptic Signaling by Postsynaptic Synapse-Associated Protein 97
J. Neurosci. Regalado et al.
26: 2343
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Point mutations in the SAP97 PDZ domains disrupt PDZ ligand binding. A, The 9-mer peptide tails of the c-termini of NR2B and CRIPT which were fused to GST. B, Representative pulldown analysis of the binding of GST-fused 9-mer c-terminal tails of NR2B or CRIPT to GFP-tagged wildtype SAP97 versus double and triple point mutants of the PDZ domains. Both GST fusion peptides bound to wildtype GFP-SAP97. In contrast, the double point mutant GFP-SAP97PDZ1&2FH was effective in preventing binding to the NR2B tail and the triple point mutant GFP-SAP97PDZ123FH prevented binding to both the NR2B tail and CRIPT-tail. GST fusion proteins (5 µg) were immobilized on glutathione-Sepharose and incubated with cell lysates of HEK293 cells expressing the indicated GFP-SAP97 polypeptide. The bound proteins and sample input were subjected to SDS-PAGE and immunoblotted using antibodies against GFP. Coomassie gels verified that equivalent amounts of GST proteins associated with the beads in each experimental sample (data not shown). This assay was replicated a minimum of 5 times for both the GST-NR2B tail and the GST-CRIPT tail.
