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. 2007 Dec;171(6):1800–1810. doi: 10.2353/ajpath.2007.070261

Figure 6.

Figure 6

Involvement of PDZ-binding motif in the interaction between RA175C and Par-3. A: Immunoprecipitation analysis for interaction between RA175C or RA175ΔC lacking EYFI and Par-3. pEB-GST-RA175C (left) or pEB-GST-RA175ΔC (right) was co-transfected with or without SRHis-Par-3 in COS cells. The cell lysate was immunoprecipitated by anti-His or anti-GST. GST-RA175C or -RA175ΔC in the anti-His immunoprecipitates and His-Par-3 in the anti-GST immunoprecipitate were detected by immunoblot analysis using anti-GST and anti-His, respectively. A complex of RA175C and Par-3 was detected in the immunoprecipitate, but a complex of RA175ΔC and Par-3 was not. B: Pull-down assay for involvement of EYFI in the direct interaction between RA175C and Par-3. MBP-RA175C or MBP-RA175ΔC were incubated with GST-Par-3 (PDZ1, 2, 3)-conjugated Sepharose beads. The bound RA175 or RA175ΔC was examined by immunoblot analysis using anti-MBP. C: Pull-down assay using RA175C-terminal peptide. Left: Direct interaction between RA175C-terminal peptide and Par-3. GST-Par-3 (1, 2, 3) was incubated with Sepharose-beads (lane 1) and RA175C-terminal peptide-conjugated Sepharose beads (lane 2). Right: The domain of Par-3 interacting with RA175C-terminal peptide. The GST-PDZ (1, 2) (lane 3), -PDZ (2, 3) (lane 4), and -PDZ (2) (lane 5) were incubated with RA175C peptide-conjugated Sepharose beads. The bound Par-3 and its domains were detected by immunoblot analysis using anti-GST.