The Journal of Neuroscience, January 26, 2005, ():
A Novel Substrate of Receptor Tyrosine Phosphatase PTPRO Is Required for Nerve Growth Factor-Induced Process Outgrowth
J. Neurosci. Chen and Bixby
25: 880
Supplemental data
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Supplementary Figure 1. Interaction between the NPCD Ptx domain and PTPRO in heterologous cells. A. GAL4-activation domain (AD) fusion constructs encoding the 1.1 kb NPCD fragment (the cDNA isolated in the yeast two-hybrid screen), the 0.6 kb neuronal pentraxin domain of NPCD (Ptx), the N-terminal half of the Ptx domain, and the C-terminal half of the Ptx domain, respectively, were co-transformed into yeast strain AH109 with the bait construct encoding the full-length intracellular domain of PTPRO fused with the GAL4 DNA-binding domain (DB). Interaction strength was analyzed in a standardized ?-gal assay. The conserved Ptx domain (aa 281-487) was sufficient to confer strong interaction with PTPRO, but neither half of the Ptx domain was sufficient for interaction. B. GAL4-DB fusion constructs encoding the full-length PTPRO intracellular domain, the juxtamembrane domain, and the catalytic domain of PTPRO, respectively, were co-transformed into yeast with the GAL4-AD fusion construct encoding the 0.6 kb conserved Ptx domain. The catalytic domain, but not the juxtamembrane domain, was necessary and sufficient for strong interaction. C. An HA-tagged protein encoded by the 1.1 kb NPCD splice variant and a Myc-tagged intracellular domain of PTPRO were co-expressed in COS cells. Precipitation of the Myc-PTPRO co-precipitated HA-NPCD (left), and precipitation of HA-NPCD co-precipitated Myc-PTPRO (right). 1/10 of the amount of lysate used in the precipitations (1/10 input) was used to assess the relative amount of each protein co-precipitated with the other.
